Cell Imaging User Training Manual

Booking Calendar Users Guide

  1. Fill out and submit a Work Authorization form. Work Auth Form For questions about this process please contact the administrative office.

  2. All users must complete the online Laser Safety Training provided by

    EHS: https://utah.bridgeapp.com/learner/courses/2d8ad69f/enroll. Upon completion,

    email your certificate to support.cellimaging@cores.utah.edu

  3. We recommend an initial half hour project consultation with Xiang or Anton. This will

    help us ensure you obtain the best images possible from your samples. This can be

    arranged with the booking calendar. Instructions below.

  4. We require 1-2 hours of microscope training prior to solo use of the

    microscopes. Please schedule time with both the microscope you want to use and

    Xiang or Anton. This can be arranged with the booking calendar. Instructions below.

  5. We suggest that you book time with staff for the start of your first imaging session. We

    also suggest that you not book after hours imaging until you feel comfortable with the

    microscope.

  6. If you want assisted imaging instead of training, this should be booked like a training

    session, i.e., book both the microscope and staff.

  7. Booking Consultation or Training

    • Each Staff member and microscope has a calendar that shows availability that you may

      book time for your use. Note that our staff have normal consultation hours at HCI, CSC

      and HSC. For any questions regarding our calendar system please email us

      at support.cellimaging@cores.utah.edu.

    • After booking staff member’s time, check the drop down option next to ‘Process’ to

      select the training location. In the ‘Description’ or ‘Service Needed’, please include

      information about your imaging needs (e.g., consultation, live cell imaging or SP8

      Training).

    • For training or assisted imaging, also book simultaneous time on the microscope.

  8. Booking a Microscope

    • Book time with both the staff member and suggested microscope simultaneously for

      initial hardware and software training.

    • Once trained, use the calendar to book time to use the microscope. Please note the

      cancelation policy below.

  9. Building access is granted after full training is complete

    • HCI

      • Computer and building access forms will be provided

      • Submit forms per our instructions

    • HSC and CSC

      • You must then go in person with your UID card to our Administrative Office to gain card

        access to the building and rooms to which you have been granted permissions. To get

        to our Administrative Office (Skaggs Bldg 582, Room 250), enter from the South doors,

        go up the stairs to the second floor, the offices are straight down the hall.

    • SMBB

      • We will send your information to the SMBB building office.•

If you have any more questions concerning this process please contact us at support.cellimaging@cores.utah.edu.

Calendar bookings must be deleted by the user 12 hours prior to the time reserved in order to avoid billing charges. The booking time cannot be altered or deleted after this point and is non-refundable nor creditable if this time window is not met. The Cell Imaging Core is not liable if a user no longer needs their booking, if a user’s samples do not work, if a user cannot make their booking, etc. However, if an instrument is down, is offline, or is under maintenance all fees will be voided for that duration.

Users with overages or caught with unregistered time can be charged $100/h.

Policies

The rules for using the Cell Imaging Core and for specific instruments.

Policies

General Policies

Calendar

It is your responsibility to reserve the time you use. You must report usage variations (overages and cancellations) to Dr. Wang before the end of the month to reconcile charges. In order for your account to work it needs to have a valid chartfield assigned to it and the core office needs to approve. If you get an error about chartfields it means yours has expired or there isn’t a valid chartfield assigned to your account. The core offices (801-581-2425) can reauthorize your account should this occur.

Usage Policies

There are limits to the number of hours a lab or user may reserve at any given time (20 business hours per week total). Exceptions must be arranged with the imaging core committee. Users who need after hours access need to contact the Cell Imaging Team about access and sign a security form in the main office RM 5C124 of the School of Medicine.

Imaging Core Advisory Committee

The committee was established July 2011 and meets about every other month to discuss various issues including upgrades to equipment, problems with equipment, allocating confocal time to large scan projects requiring >24 hour scans and service priorities for the future. The most recent meeting date is listed in the news section for the core.

Enforcement/Compliance

Users will be notified by Dr. Wang when they have violated posted core policies (oil on objectives, disregarding requests, logbook issues, not reserving time etc.) via email. The first offense they will receive email warning cc’d to the PI with a suggested remedy and a notice of the penalty schedule. Notice of the second offense will be sent by the oversight committee and copied to the PI with an invoice for a $250.00 penalty. The third notice will result in a $1000 penalty with review by the oversight committee for blocking future usage privileges. Users found without reservations will be charged the full rate plus be put on the list for a $250 fine for the second notice.

After Hours Usage

The new policy on after hours usage requires users to sign up for time in advance. Incidents of unregistered usage will be charged to the PI at $100/hr. No property of the core facility may leave the building without prior approval from core staff even for short experiments. If there is a problem in the facility after hours please contact the Cell Imaging Team.

Data Storage Issues

Our current data storage policy has been adopted to ensure that enough free space is available on hard drives for capture of new data.  The Cell Imaging core is not responsible for storing your data. We actively delete data that is more than 90 days old. We are not responsible for data lost due to hard drive failures even within the 90 day period following creation dates. Acquisition instruments are not on the network to preserve security and reduce interference with the instruments. If you need a better data transfer solution please contact the staff for advice rather than leaving your only copy of data at the facility.

Locations

Maps to the microscopes!  The Cell Imaging Core maintains microscopes in several buildings across campus.

HSC Core Research Facility

Building: 585

Code HS CORE

Map: https://cores.utah.edu/wp-content/uploads/2023/11/CellImaging-HSC-585.pdf

HCI - Cancer Research North

Building: 555

Code: HCI North

Map: https://cores.utah.edu/wp-content/uploads/2023/11/CellImaging-HCI-555.pdf

Jones Medical Research

Building: 565

Code: EEJMRB

Map: https://cores.utah.edu/wp-content/uploads/2023/11/CellImaging-EEJ-565.pdf

Crocker Science Center

Building: 5

Code: CSC

Map: https://cores.utah.edu/wp-content/uploads/2023/11/CellImaging-CSC-5.pdf

ASB - Skaggs Biology Building

Building: 82

Code: ASB

Map: https://cores.utah.edu/wp-content/uploads/2023/11/CellImaging-ASB-82.pdf

Skaggs Hall

Building: 582

Code: SK H

Map: https://cores.utah.edu/wp-content/uploads/2023/11/CoresAdmin-582.pdf

 

 

Instrument Types

Different microscopes have different uses.

 

Wide Field Microscope

Wide field microscopes illuminate the whole field of view at one time.  Imaging is fast, but out of plane information is included as blur.  There are computational methods that can reduce, but not eliminate the blur (e.g., deconvolution and extended depth of focus).

 

The Cell Imaging Core has wide field imaging systems.  These include:

Confocal Microscope

Laser Scanning Confocal

A scanning confocal illuminates one spot at a time and scans across the field of view.  There is also a pinhole to limit out of focal plane blur.  The result is a sharper image.  Further, they can be used to image the sample in 3 dimensions.  This does come at the expense of a much longer imaging time than wide field.

Spinning Disk Confocal

A spinning disk confocal uses several pinholes to gather data from multiple spots at one time.  Like a wide field, the spinning disk confocal uses a camera instead of a single point detector.  These two changes greatly reduce the imaging time, making them suitable for live cell imaging.

Multi-Photon

2-photon/multi-photon microscopes rely on 2 or more simultaneous lower energy photons to stimulate fluorescence.  One of the main advantages of this method is that the lasers can penetrate deeper into the sample.  It does require a more powerful laser and working in the IR.  Applications include samples up to a few mm thick and intravital microscopy.

Super Resolution

Resolution in microscopy is diffraction limited to roughly half the wavelength of the light being detected.  Super resolution methods use additional information to break through the diffraction limit.  This can be extra spatial or temporal information and may require additional data collection as well as additional computation to generate the high resolution image.

Airy Scan

In an Airy scan microscope, multiple detectors are used to interrogate the spatial blur or Airy pattern.  This additional information is then used to reduce the blur, achieving higher resolution than normal confocal.

STED

STimulated Emission Depletion uses a laser to deplete emission around the focal spot.  This provides much higher lateral resolution.  This is one of a few super-resolution microscopy methods.

Structured Light

If several images are made with known patterns of light, the images can be combined to create a higher resolution image.

The Nikon Spinning Disk uses structured light to in its LiveSR unit.

TIRF

Total Internal Reflection Fluorescence microscopy achieves thin Z sectioning at the expense of only being able to image the first thin (<200nm) layer of the sample.  It does have a high signal to noise ratio because there is very little out of plane fluorescence.  This makes the method useful for for events in cellular surfaces.

 

 

 

Instrument Details

Details about each instrument

Instrument Details

Zeiss 700 Confocal Microscope

Location: HSC (Building 586) Room 56

image-1700073367695.jpeg

The LSM 700 laser scanning confocal microscope is the seventh generation of confocal microscopes from Carl Zeiss. The LSM 700 system uses a Zeiss AXIO Observer Z1 inverted microscope with transmitted illumination (HAL 100) and a laser illumination source. The LSM 700 system features easy operation, excellent sensitivity, and a design that can handle complex tasks.

Main features of Zeiss 700 System:

Suggested Applications:

Imaging lasers:

Wavelength (nm) Type Power Manufacturer
405 diode 5 mW

Lasos Lasertechnik GmbH
488 diode 10 mW

Lasos Lasertechnik GmbH
555 diode 10 mW

Lasos Lasertechnik GmbH
639 diode 5 mW

Lasos Lasertechnik GmbH

Objectives:

Objective Magnification Immersion

Numerical

Apperature

 Correction Ring Coverglass (mm)

Working

Distance (mm)
EC Plan Neofluar M27 10X Air 0.3
0.17

5.2
Pan Apo M27 20X Air 0.8
0.17 0.55
LD C-Apo Korr M27 40X Water 1.1 Corr 0.14-0.19 0.62
Pan Apo DIC M27 63X Oil 1.4
0.17 0.19

Instrument Details

Prairie Ultima 2

Location: HSC Room 48B / 48D

Main features of Prairie Ultima 2/Multi-Photon Confocal System:

Imaging lasers:

Suggested Applications:

Objectives:

48B:
Objective Magnification Immersion Numerical Aperture Correction Collar Coverslip (mm) Working Distance (mm)
Apo 25 Water, Water Dipping 1.1 Corr 0-0.17 2
48D:
Objective Magnification Immersion Numerical Aperture Correction Collar Coverslip (mm) Working Distance (mm)
Plan Apo Lambda 4 Air 0.2

20
Plan Fluor 10 Air 0.3
0.17

16
UPlanFl 10 Air 0.3


LWD 16 Water Dipping 0.8
0

3
Apo 25 Water, Water Dipping 1.1 Corr 0-0.17

2
Fluor 40 Water Dipping 0.8
0

2

Instrument Details

Nikon A1R Confocal

Location: HSC Room 56

Main features of System:

Imaging lasers:

Suggested Applications:

Objectives:

Objective Magnification Immersion Numerical Aperture Correction Ring Coverglass Working Distance
Plan Apo Lambda 4 Air 0.2

20
Plan Apo Lambda 10 Air 0.45
0.17 4
Plan Apo Lambda 20 Air 0.75
0.17 1
Plan Fluor 40 Oil 1.3
0.17 0.2
Plan Apo Lambda 60 Oil 1.4
0.17 0.13

Other objectives available. Please inquire with Core personnel

Instrument Details

Evos Auto Color and Fluor Microscope

Location: HSC (Building 586) Room 54

CI-Evos.jpg

The EVOS FL Imaging system is an all-in-one fluorescence microscope designed for efficiency and ease of use. The on-board computer and integrated imaging software allows users to capture and save four channels fluorescence images/data directly from the microscope. The EVOS FL Imaging System is suitable for checking samples before using a high-end microscope system.

Main features of System:

Suggested Applications:

Light Sources:

LED light cubes which combine bright LED illumination with excitation and emission filters.  DAPI, CY5, GFP, RFP and Texas Red cubes are available.  Four can be installed at one time.  Contact Cell Imaging staff to change cubes if needed.  A selection guide for matching cubes to dyes is on the ThermoFisher site: Guide

Cube Excitation (nm) Emission (nm)
DAPI 357/44 447/60
GFP 485/25 524/24
RFP 531/40 593/40
Texas Red 585/29 628/32
Cy5 628/40 692/40

Objectives:

The objectives on the Evos are Long Working Distance, not Coverslip-Corrected.  They are optimized for use through vessels with a nominal wall thickness of 0.9-1.5 mm.

Objective Magnification Immersion

Numerical

Apperature

 Correction Ring Coverglass (mm)

Working

Distance (mm)
PlanApo 1.25X Air 0.04

5.10
Plan Fluor 4X Air 0.13

17.2
Plan Fluor 10X Air 0.3
1.2 8.31
Plan Fluor 20X Air 0.45
1.2 7.11
Plan Fluor 40X Air 0.65
1.2 2.81


Instrument Details

Zeiss Axio Scan.Z1

Location: HSC (Building 586) Room 60

CI-ZeissAxioScanZ1.jpg

The Zeiss Axio-Scan.Z1 is a fast and flexible slide scanner that digitizes specimens to produce high-quality virtual slides. In addition to brightfield slide scanning, the Axio-Scan.Z1 uses LED lights to scan up to nine fluorescent channels. Up to 100 slides can be loaded onto the scanner, making it suitable for large projects, including digital archives. Scanned images are saved as CZI files and can be viewed using Zeiss freeware Zen-lite or third-party image analysis programs.

Main features of Axio Scan.Z1:

Light Sources:

In addition to an internal lamp for brightfield imaging, the Axio Scan.Z1 has an X-Cite 120LED source for fluorescent imaging.

Filter cubes for LEDs:

The scanner has a 10 position filter turret to fit most fluorophores.  The Zen profile wizard is used to pick filter to match your imaging needs.  Alternate filter cubes can be installed for specialty applications.  Please consult with Cell Imaging staff for consultation and assistance.

Filter Position Excitation (nm) Emission (nm) Typical Fluorophores Opal Dye
1 empty empty Brightfield
2 325-375 435-485 DAPI
3 426-446 465-495 Cyan, Protein 480
4 480-500 510-530 Alexa Fluor 488 520
5 510-530 540-560 Alexa Fluor 489 540
6 538-562 570-640 HE, DsRed, Alexa Fluor 555 570
7 568-592 610-640 Alexa Fluor 568 620
8 610-630 645-675 Alexa Fluor 660 650
9 625-655 666-715 Cy5
10 650-680 695-755 Alexa Fluor 680 690

Objectives:

Objective Magnification Immersion

Numerical

Apperature

 Correction Ring Coverglass (mm)

Working

Distance (mm)
EC Plan-Neofluar M27 2.5X Air

0.085
0.17

8.8
Fluor M27 2.5X Air

0.12
0.17

8.7
Fluar M27 5X Air 0.25
0.17

12.5
Plan Apo M27 10X Air 0.45
0.17

2.1
Plan Apo M27 20X Air 0.8
0.17 0.55
Plan Apo Korr M27 40X Air 0.95
0.13-0.21 0.25

 

 

Instrument Details

Nikon Automated Widefield Microscope

Location: HCI Room 1430

Main features of System:

Suggested Applications:

Objectives:

Objective Magnification Immersion Numerical Aperture Correction Ring Coverglass (mm) Working Distance (mm)
Plan Apo Lambda 4 Air 0.2

20
Plan Apo Lambda 10 Air 0.45

0.17

 4
Plan Apo Lambda 20 Air 0.75
0.17 1
S PLAN Fluor ADM ELWD 20 Air 0.45 Corr 0-2.0 8.2-6.9
Plan Apo Lambda 60 Oil 1.4
0.17 0.13
Plan Apo Lambda 100 Oil 1.45
0.17 0.13

Other objectives available. Please inquire with Core personnel

Instrument Details

Leica SP8 White Light System

Location: HCI Room 1480

Main features of Leica SP8 White Light System:

Imaging lasers:

Suggested Applications:

Objectives:

Objective Magnification Immersion Numerical Aperture Correction Ring Coverglass (mm) Working Distance (mm)
HC PL APO CS2 10 Air 0.4

2.74
HC PL APO CS2 20 Either water, glycerine, or oil 0.75 Corr
0.66
HC PL APO CS2 20 Air 0.75
0.17 0.62
HC PL APO CS2 40 Water 1.1 Corr 0.14-0.18 0.65
HC PL APO CS2 40 Oil 1.3
0.17 0.24
HC PL APO CS2 60 Oil 1.4
0.17 0.14

Other objectives available. Please inquire with Core personnel

Instrument Details

Leica SP8 405-488-561-633 Laser Confocal

Location: HCI Room 1440

Main features of Leica SP8 White Light System:

Imaging lasers:

Suggested Applications:

Objectives:

Objective Magnification Immersion Numerical Aperture Correction Collar Coverslip (mm) Working Distance (mm)
HC PL APO CS2 10 Air 0.4

2.74
HC PL APO CS2 20 Either water, glycerine, or oil 0.75 Corr
0.66
HC PL APO CS2 20 Air 0.75
0.17 0.62
HC PL APO CS2 40 Water 1.1 Corr 0.14-0.18 0.65
HC PL APO CS2 40 Oil 1.3
0.17 0.24
HC PL APO CS2 63 Oil 1.4
0.17 0.14

Instrument Details

Zeiss Axioscan 7

Location: HCI Room 1470

CI-Axio7.jpg

The Zeiss AxioScan 7 is a fast and flexible slide scanner that digitizes specimens to produce high-quality virtual slides. In addition to brightfield slide scanning, the Axioscan 7 can scan up to five fluorescent channels using fast switching LED lights. Up to 100 slides can be loaded onto the scanner, making it suitable for large projects, including digital archives. Scanned images are saved as CZI files and can be viewed using Zeiss freeware Zen-lite or third-party image analysis programs.

Main Features of the Axioscan 7:

Suggested Applications:

Configurations:

The Axioscan 7 has a replaceable filter turret and can be switched between two light sources.  The 10 position filter cube turret is typically used with the Colibri 7 switchable LED source.  The fast filter wheel is typically used with the X-Cite Xylis LED source.

Configuration Light Source Filter Set
1 Colibri 7 Filter Cubes
2 X-Cite Xylis Fast Filter Wheel

Only Cell Imaging staff are permitted to switch between configurations.   Please schedule our time and plan accordingly.

 Light Sources:

Colibri 7:

The Colibri 7 has 6 LEDs with excitation filters to provide 7 excitation bands.  (One LED has a motorized filter to split it into two bands.)

Excitation Bands (There is one too many here)

Band Wavelength / Bandwidth (nm) Some typical fluorophores
UV 385/30

API, Hoechst 33342, Hoechst 33258, Alexa Fluor 350, Alexa Fluor 405, Indo-1, eBFP / BFP, eGFP (wt), True Blue
V 423/44

Pacific Blue, Lucifer Yellow, Alexa Fluor 433, eCFP, Cerulean
B 469/38

FM1-43, Cy2, eGFP, NBD, MitoTracker Green, Alexa Fluor 488, BCECF, Calcein, DiO SNAFL, YO-Pro-1, Nissl, LysoSensor Green, mHoneydew, FITC / Fluorescein, Kaede (green / red), PerCP, YoYo-1,FuraRed
C 511/44

Rhodamine 123, Fluo-4, Oregon Green BAPTA, Sytox Green, eYFP, FM4-64, Eosin/HE, Acridine Orange, JC1, Bodipy FL, Propidium Iodide, Spectrum Green, Calcium Green
G 555/30

TRITC, 7-AAD, Cy3, tdTomato, Alexa Fluor 546, Alexa Fluor 555, DsRed, mOrange, TagRFP, SNARF, DyLight 549, Spectrum Orange
Y 590/27

MitoTracker RED FM/CMXRos, txRed, mCherry, mRFP1, Cy3.5, Rhodamine B, Alexa Fluor 568, Dylight 594, Alexa Fluor 594, Bodipy TR
R 631/33

Alexa Fluor 633, Alexa Fluor 647, Cy5, DRAQ5, ToTo-3, ATTO-655, MitoTracker DeepRed, APC, ATTO-647N
FR 735/40

Alexa Fluor 750, Alexa Fluor 790, Cy7, Cy7.5
X-Cite Xylis:

The X-Cite Xylis XT720L is a broad spectrum illuminator with a range from 380-770nm.  It has peaks at 385, 430, 475, 545, 635, 735nm.

Filters:

Filter Cubes:

In multi-band fluorescence imaging, the filter turret has to move between positions for each band.  This is usually done on a per-tile basis.  At about 400ms per move, this can add up, especially for a large scan at higher magnification.  The Axioscan 7 has 2 multi-band cubes.  If these can be used, it can reduce or eliminate these moves and reduce scan time.  These can be used on the Axioscan 7 because it has the Colibri 7 switching LED source.

Filter No

 Fluorochrome

Excitation (nm)

Beamsplitter (nm)

Emission (nm)

1

Empty (Brightfield)

 

 

 

2

HE GFP

450-490

495

500-550

3

HE DsRed

538-562

570

570-640

4

Cy5

625-655

660

665-715

5

HE BFP

370-410

420

430-470

6

HE DAPI/GFP/Cy3.5/Cy7

375-395

455-483

583-600

720-750

405

493

611

762

410-440

501-547

618-650

770-800

7

HE DAPI/GFP/Cy3.5/Cy5/Cy7

370-400

450-488

540-570

614-647

720-750

405

493

575

654

761

412-438

501-527

582-601

662-700

770-800

Pol

Circular polarizer and analyzer




ND

6% Neutral Density Filter




Fast Filter Wheel:

The fast filter wheel moves between filter positions about 10 times faster than the filter cube turret.

Need information here

Filter Pass Peak and Width Typical fluorophores
Empty (Brightfield)

Single Channel 1

Single Channel 2

Single Channel 3

Dual Channel 4

Dual Channel 5

Objectives:

Objective Magnification Immersion

Numerical

Apperature

 Correction Ring Coverglass (mm)

Working

Distance (mm)
Fluar M27 5X Air 0.25
0.17

12.5
Pan Apo M27 10X Air 0.45
0.17 2.1
Pan Apo M27 20X Air 0.8
0.17 0.55
Pan Apo Korr M27 40X Air 0.95
0.13-0.21 0.25
EC Pan-Neofluar Pol M27 20X Air 0.5
0.17 2


Instrument Details

Zeiss 880 Airy Scan

Location:CSC Room 032

CI-Zeiss880.jpeg

Main features of Zeiss 880 Airy Scan System:

Imaging lasers:

Suggested Applications:

Objectives:

Objective

 Magnification Immersion Numerical Apperature Correction Ring Coverglass (mm) Working Distance (mm)
Plan Apo M27 5 Air 0.16
0.17 12.1
EC Plan-Neofluar M27 10 Air 0.3
0.17 5.2
Plan Apo M27 20 Air 0.8
0.17 0.55
Plan Apo M27 40 Oil 1.3
0.17 0.21
Plan Apo DIC M27 63 Oil 1.4
0.17 0.19
Calibration Apo DIC M27
 




Instrument Details

StedyCon

Location: CSC Room 30

Main features of StedyCon:

Objectives:

Objective Magnification Immersion  Numerical Apperature Correction Ring Coverglass (mm) Working Distance (mm)
Plan Apo Lambda 4X Air 0.2

20
Okan-Neofluar 5X Air 0.15
0.17 13.6
Plan Apo 10X Air 0.45   0.17 4
PlanApo Lambda 20X Air 0.75
0.17 1
Plan Apo Lambda 60X Oil 1.4
0.17 0.13
Plan Apo Lambda 100x Oil 1.45
0.17 0.13

 

Instrument Details

Olympus FV1000 Confocal Microscope

Location: CSC Room 32 

Main features of System:

Imaging lasers:

Objectives:

Objective Magnification Immersion  Numerical Apperature Correction Ring Coverglass (mm) Working Distance (mm)
UPlanSApo 10X Air 0.4
0.17 3.1
UPlanApo 20X Air 0.7
0.17 0.65
UApo 40X Oil 1.35 Corr 0.17 0.1
PlanApo 60X Water 1.2 Corr 0.13-0.21 0.28
PlanApo 60X Oil 1.4
0.17 0.12


Instrument Details

Delta Vision Widefield

Location: EEJMRB Room 5100

CI-Deltavision.png

Hardware:

Primary applications:

Objectives:

Objective Magnification Immersion Numerical Aperture Correction Ring Coverslip (mm) Working Distance (mm)
UPlanXApo 4 Air 0.16

13
UPlanXApo 10 Air 0.4
0.17

3.1
UPlanXApo 20 Air 0.8
0.17

0.6
UPlanXApo 40 Oil 1.4
0.17

0.12
UPlanXApo 60 Oil 1.42
0.17

0.15


Instrument Details

Leica Spinning Disk Confocal

Location: EEJMRB Room 5122

Hardware:

Primary applications:

Objectives:

Objective Magnification Immersion Numerical Aperture Correction Ring Coverglass (mm) Working Distance (mm)
HC PL APO CS2 10 Air 0.4

2.74
HCX PL APO CS 20 Air 0.75
0.17 0.62
HC PL APO CS2 40 Water 1.1 Corr 0.14-0.18 0.65
HC PL APO CS2 63 Oil 1.4
0.17 0.14


Instrument Details

Nikon Ring TIRF or Spinning Disk Confocal

Location: EEJMRB Room 3532


Objective Magnification Immersion Numerical Apperature Correction Ring Coverglass (mm) Working Distance (mm)
Plan Fluor 10 Air 0.3
0.17 16
Plan Fluor 10 Air 0.3
0.17 16
Plan Apo 20 Air 0.75
0.17 1
Apo TIRF 60 Oil 1.49 Corr 0.13-0.19 0.16-0.07
Plan Apo Lambda 60 Oil 1.49
0.17 0.13
Apo TIRF 100 Oil 1.49 Corr 0.13-0.19 0.16-0.09

 


Image Analysis Software

Imaris, Nikon Elements, and Zen.  And some recommendations for other solutions.

Image Analysis Software

Overview

With our microscopes you can generate large complex multidimensional data sets.  The analysis tools have to be powerful enough to deal with the data.  While there is good open software for some applications, there are data sets where you may need commercial software and more powerful workstations.

Workstations: HSC Room 60, HCI Room 1470

The Cell Imaging core has two image analysis workstations for your use.  These workstations have many large hard drives to accommodate image data sets.  Graphics cards are installed for faster analysis.  Commercial and open image processing software is installed.  The workstations and software are available for reservation through the Resource Calendar.

Imaris

Imaris is made for 2D, 3D and 4D image visualization and analysis of multichannel fluorescence images.  It can handle large data sets, extract objects, make measurements, and make graphics and movies for presentations and publication.

Strengths:

We have a one-seat floating license for this software.  Please reserve time on the software in the Resource Calendar.

We have additional week long licenses available for rent for use on your own workstation.  Please inquire with the Cell Imaging staff for more information. 

A free image viewer is available for Window or Macintosh. Imaris Viewer.

Nikon Elements AR ai

Nikon Elements provides 2D, 3D and 4D image visualization of multichannel images.  It provides a large set of tools for image manipulation, analysis and measurement.  Analysis programs can be written as macros (a C like language) or using the General Analysis tool (a graphical programming environment).  These can then be applied in batch across many images.

Strengths:

We have a one-seat floating license for this software.  Please reserve time on the software in the Resource Calendar.

A free image viewer is available for Windows or Macintosh. NIS Viewer.

Zeiss Zen Desk (only at HCI 1470)

Zen provides several image visualization, image analysis, and measurement tools.

Strengths:

We have one license for this software.  It is only available at HCI 1470.

A free image viewer is available for Windows. Zen-Lite. Zen-Lite also provides some image processing tools and is especially useful for image export to other formats.  

First Steps - Training Procedure

Introduce rules/guidelines. 

How to access the building, computer, and software; how to book the calendar; follow the SOPs; how to seek help; communication between users.

Introduce three portions of the whole imaging system. 

Bulky hardware could be a power supply, control box, lasers, laser power supplies, xyz stage controller.  The user should know the order of how to turn it on/off.  Advanced: to recognize each box/part.

The second part is the mainframe of the microscope.  Start from the Light sources, show the light path to tell upright or inverted configuration so they know how to put the sample on correctly; how many detection locations (usually four: three ports from microscope body and one is for eyepiece); main UNTOUCHED box, where the major optical components are located;  how many objectives (air, water or oil immersion, advanced knowledge (features of the objective, working distance, NA, Correction…)); how many lasers,  wavelength covered and how to switch channels (advanced knowledge: pulse or CW, solid-state or gas, single line or multiple lines, output energy, life span…); Epifluorescence light and how to switch channels;  the right position to put samples on the stage; how to move the stage (xyz, coarse and fine); how to see the image and switch between the eye and detector; how to put a water/oil on the objective and clean the objective after you have done

The third part is the software on the computer to acquire data and transfer data. 

Introduction of the software (the introduction is based on the Olympus Fluoview)

·       Set up an individual account. 

Log in as the Administrator, set up an individual account as “guest”?

The username format in each account is “User’s Firstname_PI last name”. The password is random.  User/trainer should write this down.

·       Log into a new account.

The first window (explorer):

Point out the “Jump to…” Icon

       The second window (Data)

Do a quick live scan and show the data information.  Introduce the memory issue if data was left in the memory

        The Third Window ( Setting)

·       Scanning model

·       Scanning shape

·       Speed, pay attention to the unit. So, a user will understand the number they are using.  Explain the pros and cons why use different values

·       Time indication. L, F, S

·       Resolution (512x512 for general, >=1024X1024 for publication, pros and cons)

·       Aspect ratio

·       Rotation

·       Zoom (digital zoom vs real resolution change)

·       Set laser power

·       Lambda measurement (don’t teach)

·       Z Stack  drop down menu for objective selection; while running the live imaging, click the arrow to move the objective to the top, click “set”, then move to the opposite move direction, and “go” button; move back to the middle and make sure no saturation; high-low mode, ctrl+h; select Z step (z resolution)

·       Time lapse (can mention, don’t teach the detail)

The Fourth window (acquire)

·       Select three light sources to observe the sample.

·       For fluorescence dyes, how to add/remove channels

·       The next column can briefly introduce them.

·       To acquire, explain FocusX2 and why; explain XY repeat; explain “Auto HV” back to “Speed” at the third window; use XY (ZTL) to take a final imagine;

·       Channels (HV, Gain and Offset)

·       Pinhole size

·       Transmit light intensity

·       Save file to Hard drive

Hands-on and introduction to the Fifth window (Live view)

·       Left: how to select window and channel

·       Single window vs multiple windows

·       Merge channels

·       1:1 vs Full window

Save file

·       Format of the raw data (*.oib for 2D ; *.oij for 3D)

·       Export data

·       “Save display” function and the trick

·       “Add a view” function

Play move

Do/undo Z projection and save

To browse data back to office, Olympus Fluoview viewer (link) and ImageJ (link)

Transfer data by using USB portable device

Cleaning up the water or oil on objectives.

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