ReadPlate

Author:

Jose Maria Delfino (delfino@qb.ffyb.uba.ar)

Department of Biological Chemistry and Institute of Biochemistry and Biophysics (IQUIFIB)

School of Pharmacy & Biochemistry, University of Buenos Aires and CONICET

Junin 956, C1113AAD Buenos Aires, Argentina

Phone: 54 11 4962 5506, extension 116

History:

August 15, 2020: ReadPlate 3.0 version incorporates the capability of reading multi-well plates

of 6 (3x2), 12 (4x3), 24 (6x4), 48 (8x6) or 96 (12x8) wells. A new user-friendly interface

facilitates image analysis. This version allows new user-defined features to optimize blank

correction. Multiple readings of the same plate have also considerably been facilitated, appending

the full parameter file to the results and log file

February 16, 2018: ReadPlate2.1 incorporates an improved blank correction algorithm

April 15, 2016: ReadPlate2 introduces a correction for blank measurements

March 10, 2016: Inclusion of alpha numeric labeling for the wells and calculation

of absorbance values

December 16, 2015: Original version

With thanks to Sandra Verstraeten, Pablo Carabias, Irene Mangialavori and Gabriela Gomez for

their enthusiasm, suggestions and help with this project

Key words:

Plate reader, microplate reader, microtiter plate photometry, absorbance, digital camera, cell-phone camera

Relevant literature reference:

Carla R. Angelani, Pablo Carabias, Karen M. Cruz, Jose M. Delfino, Marilina de Sautu,

Maria V. Espelt, Mariela S. Ferreira-Gomes, Gabriela E. Gomez, Irene C. Mangialavori,

Malena Manzi, Maria F. Pignataro, Nicolas A. Saffioti, Damiana M. Salvatierra Frechou,

Javier Santos, Pablo J. Schwarzbaum

"A Metabolic Control Analysis Approach to Introduce the Study of Systems in Biochemistry:

the Glycolytic Pathway in the Red Blood Cell"

Biochemistry and Molecular Biology Education, Volume 46, Issue5, September/October 2018,

Pages 502-515

https://doi.org/10.1002/bmb.21139

Source:

The source code of ReadPlate3.0 version is available at

../../macros/ReadPlate3.0.txt

Installation:

Download ReadPlate3.0.txt and do Plugins > Install. The plugin is ready to be launched by

clicking Plugins >ReadPlate3.0

An example image of a 96-well plate is available at

../../macros/images/plate.jpg

Description:

Image acquisition:

The multi-well plate is located on top of a home-built trans-illuminator: a white 7 x 10 LED array,

base (Figure A). To avoid the influence of stray light, the device is covered with a tall black

plywood pyramidal box equipped with a central hole at the top (Figure B), through which the

zoom lens of the camera is located (Nikon CoolPix S6300 in our case, but cell-phone cameras can be

used as well). The vertical optical axis passes through the center of the plate. To minimize parallax

error that would affect the light path through the samples, pictures are taken at a minimal distance

of 70 cm. The plate borders should be parallel to the frame of the picture. The example

photograph (stored as a .jpg file) corresponds to a 96-well plate with samples of a colorimetric

assay for lactate (120 μL per well, maximum of absorbance at 555 nm). In this case, the green

channel shows the highest sensitivity, due to maximal spectral overlap between the absorption

spectrum of the chromophore in the sample and the green window.

Plugin use:

Before starting (very important!), please set the following parameters for measurements:

(Analyze > Set Measurements). The following should be selected: Area / Standard Deviation /

Min & Max Gray Value/ Mean Gray Value/ Modal Gray Value/ Add to Overlay / Redirect to None /

Decimal Places (0-9): 3. ReadPlate measures the color intensity (RGB) of an image (.jpg file) of a

multi-well plate of 6 (3x2), 12 (4x3), 24 (6x4), 48 (8x6) or 96 (12x8) wells. The color photograph

of the plate should be centered at the middle point. For further details and validation against a

commercial plate reader, see the Supplementary Material (Figure S3) of the literature reference

cited above. Open the .jpg image from within the ImageJ software (tested with version 1.53c,

Wayne Rasband, NIH, 26 June 2020). Run the plugin ReadPlate (by launching Plugins > ReadPlate).

Select the correct plate format: 6(3x2), 12(4x3), 24(6x4), 48(8x6) or 96(12x8) wells (Figure C).

Make a center-to-center rectangular selection of wells (upper-left and lower-right corners, Figure D).

Select the desired color channel (Red, Green, Blue or Gray) for measurements (Figure E). Choose

the appropriate parameters for the grid of circles to be measured (Figure F). Each circle needs

not be too large, because enough color information is coded in pixels covering a relatively small

area. The final grid overlaid on the plate should show main circles centered on each well surrounded

by ancillary circles located outside (Figure G). The latter are used by the blank correction

algorithm to compensate for any difference in local light intensity (Figure H). Check the fit of the

final grid onto the plate image. If satisfactory, the user proceeds to collect measurements.

A table of results will appear next (Figure I). This includes number, alphanumeric

label and area of each well, light intensity measurements (Mean, StdDev, Mode, Min, Max),

and absorbance values. Corrected absorbance values (Acorr) result from applying the blank

suppression algorithm. Note that all measurements are taken on a single image of the plate, thus

eliminating the need for taking parallel measurements on an empty plate.

By default, results are formatted as .csv files, readily interpretable by Excel (Figure J).

A - The trans-illuminator

B - The black box and cameras

C - Choosing the plate format

D - Making the rectangular selection

E - Selecting the color channel

F - Setting the parameters

G - Checking the fit of the grid

H - The blank suppression procedure

I - Getting results in tabular form

J - Exporting results in Excel format