Introducing: Cytation™3 Cell Imaging Multi-Mode Reader

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

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

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

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

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

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

Optical Path

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

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

Filter Cubes

Slider with 4 cube positions

LED cube with color LEDs

LED cube in place

Filter Cube: Excitation and Emission Filter + Dichroic

Filter Cube Sits on Top of LED Cube

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…

Objectives

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

Magnification vs. well size 2x 2.5x 4x 10x 20x 96-well 6.5 mm 384-well 3.7 mm

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

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

Objective adapters

Reading slides, reading microplates

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

Numerical aperture (NA) and Working Distance

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…

CCD camera

Compact grayscale CCD camera

16-bit Sony CCD Chip

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

CCD Camera Mounted in Instrument

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.

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

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 = 4095 values (0=black, 4095 = white) 16-bit = 65535 values (0=black, 65535 = white)

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 65535 is considered “saturated” (equivalent to “overflow” on PMT), but pixel value will still be set to 65535 (software will have ability to identify and highlight saturated pixels).

Artificial Color in Gen5