ReadPlate

 

Author:

Jose Maria Delfino (delfino at 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:

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 and Gabriela Gomez for their enthusiasm,

suggestions and help with this project

 

Source:

The source code of ReadPlate2.1 version is available at

../../macros/ReadPlate2.1.txt

 

Installation:

Download ReadPlate2.1.txt and do Plugins > Install. The plugin is ready to be launched by

clicking Plugins >ReadPlate2.1

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,

powered by a 12V DC power supply, covered with an acrylic plate that acts as a light-diffusing

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 (Nikon CoolPix S6300 in our case, but also cell-phone cameras can be

used) is located. The vertical optical axis passes through the center of the plate (the D6/E7

point). To minimize the parallax error that would affect the light path through the samples,

pictures are taken at a least distance of 70 cm. The plate borders should be parallel to the frame

of the picture. Figure C is an example photograph (stored as a .jpg file) of a 96-well plate with

samples of a colorimetric assay for lactate (120 μL per well, absorbance maximum at 555 nm). In

this case, the green channel shows the highest sensitivity, due to maximal spectral overlap

between the absorbance spectrum of the chromophore and the green window.

 

Plugin use:

ReadPlate measures the color intensity (RGB) of an image (.jpg file) of a multi-well plate of up to

96 wells. After choosing the appropriate color channel for measurements (Figure D), a grid of

circles of defined area is built and overlaid on the plate (Figures E and F). If the fit of the grid to

the plate proves to be satisfactory, the user proceeds to take measurements. These include the

distribution of light intensity within each central circle and the calculated absorbance values for

each well, which are listed in tabular form (Figure G). Results can be readily exported in Excel

spreadsheet format (Figure H). The software uses an ancillary grid of circles (of equivalent area,

to contain a similar number of pixels) surrounding each well to correct for differences in local

light intensity. ReadPlate2.1 introduces an improved blank correction algorithm, which removes

outlier values due to the design of the particular plastic plate used or the eventual presence of

spurious specks. In this fashion, absorbance values are corrected for blank measurements taken

from the same image of the plate, eliminating the need for taking parallel measurements on an

empty plate.

 

A –The trans-illuminator

Description: Macintosh HD:Users:jmdelfino:Desktop:ReadPlate2.1:text&images:A.jpg

 

B – The black box

Description: Macintosh HD:Users:jmdelfino:Desktop:ReadPlate2.1:text&images:B1.jpgDescription: Macintosh HD:Users:jmdelfino:Desktop:ReadPlate2.1:text&images:B2.jpg

 

C – An image of a 96-well plate

Description: Macintosh HD:Users:jmdelfino:Desktop:ReadPlate2.1:text&images:C.jpg

 

D –Selecting the color channel

Description: Macintosh HD:Users:jmdelfino:Desktop:ReadPlate2.1:text&images:D.jpg

 

E – Selecting the parameters for building the grid

Description: Macintosh HD:Users:jmdelfino:Desktop:ReadPlate2.1:text&images:E.jpg

 

F – Checking the size and position of the grid of circles

Description: Macintosh HD:Users:jmdelfino:Desktop:ReadPlate2.1:text&images:F.jpg

 

G – Getting the results in tabular form

Description: Macintosh HD:Users:jmdelfino:Desktop:ReadPlate2.1:text&images:G.jpg

 

H – Exporting the results to an Excel spreadsheet

Description: Macintosh HD:Users:jmdelfino:Desktop:ReadPlate2.1:text&images:H.jpg