1. Introducing: Cytation™3 Cell Imaging Multi-Mode Reader

2. Project Goals Main project goal was to design:
An affordable microplate imager to support increased cell biology research
A combined microplate reader / imager that provides new benefits such as a unique hit-picking feature
Easy-to-use software interface to reduce learning curve for non-imaging experts

3. Traditional Plate Reader - Filters
Quantitative data and sensitivity
Precisely measure cellular activity

4. Traditional Plate Reader - Monochromator
Quantitative data and flexibility
Precisely measure cellular activity

5. Imaging Optical Path Qualitative and semi-quantitative data
Visualize change in phenotype

6. Microplate Reader with Imaging
Quantitative numerical data
Quantitative numerical data
Fuller picture for cell biology research
Qualitative data
Qualitative phenotypic data

7. More and more assays are run on live cells
Cytation3 live cell features:
Temperature control up to 45ºC
Gas controller (CO2 / O2)
Automatic reagent dispenser
Quantitative numerical data
Qualitative phenotypic data

8. Optical Path

9. CCD Fluorescence Optical Path - Widefield 4. Samples
5. Gray scale CCD camera
3. Objective
CCD
2. Filter/mirror cube
1. Light source (LED)

10. CCD Bright Field Optical Path 1. Light source (LED) 2. Samples
4. Gray scale CCD camera
3. Objective
CCD

11. Filter Cubes

12. Slider with 4 cube positions

13. LED cube with color LEDs

14. LED cube in place

15. Filter Cube: Excitation and Emission Filter + Dichroic

16. Filter Cube Sits on Top of LED Cube

17. DAPI GFP Texas Red RFP YFP CY5 CY7 CFP Custom…
8 Colors Available Right Away
DAPI
GFP
Texas Red
RFP
YFP
CY5
CY7
CFP
Custom…

18. Objectives

19. BioTek objectives (also called “lenses”):
Zeiss 2.5 x
Olympus 4 x
Olympus 10 x
Olympus 20 x
No-brand 2x

20. Magnification vs. well size2x
2.5x 4x
10x
20x
96-well
6.5 mm
384-well
3.7 mm

21. 384-well 3.7 mm Magnification vs. pixel size 4x 2 x 1.5 mm 2.5x

22. What does 2.5x, 4x, 10x… mean? Measurement area in the well
(2.0 x 1.5 mm)
CCD sensor
(about 8 x 6 mm)
4 x
Computer monitor
(example: 40 x 30 cm)
Projection screen
(160 x 120 cm)
50 x
200 x
Objective magnification
800 x

23. Objective adapters

24. Reading slides, reading microplates

25. Numerical aperture (NA) and Working Distance
Microplate applications typically require long working distance objectives

26. Numerical aperture (NA) and Working Distance

27. Magnification and example of applications
2 x 1.5 mm
20x
0.4 x 0.3 mm
2.5x
3 x 2.3 mm
No cell details at all
Counting could be difficult (many overlapping objects)
In this case, total image intensity would be used rather than count.
Limited cell details
Allows counting, and many cells are visible so good statistics
Cytotoxicty, cell proliferation assays
Intracellular details visible
Qualitative information about location of fluorescence in cells
Advanced image analysis can be performed in Gen5 or third party software.
Apoptosis, translocation events…

28. CCD camera

29. Compact grayscale CCD camera

30. 16-bit Sony CCD Chip

31. Only One Firewire Port is Used (and Visible on the Instrument)

32. CCD Camera Mounted in Instrument

33. Why FireWire? High Data Transfer Speed.
Firewire Port Card (Laptop or Desktop)
Why FireWire? High Data Transfer Speed.
What about smaller laptop Firewire Ports? No Power.

34. Each image contains about 1,2 million pixels
CCD chip and Pixels
CCD sensor / chip
Sony chip
Number of pixels:
1384 x 1036 (sensor)
1280 x 960 (image)
Pixel size 4.65 um
Each image contains about 1,2 million pixels

35. Common camera bit depth: 8-bit = 255 values (0=black, 255 = white)
Pixel value = 0 (black)
Pixel value = 22,645 (gray)
Pixel value = 65,535 (white)
Common camera bit depth:
8-bit = 255 values (0=black, 255 = white)
12-bit = values (0=black, 4095 = white)
16-bit = values (0=black, = white)

36. Gray scale No color information is stored per pixel: gray scale image
Colors are added after the fact by Gen5 (see next slide)
A pixel at is considered “saturated” (equivalent to “overflow” on PMT), but pixel value will still be set to (software will have ability to identify and highlight saturated pixels).

37. Artificial Color in Gen5