How to benchmark a wide field 

fluorescent microscope Detection threshold (ms) Saturation (ms) Intensity response (counts/(



m2ms))

Michael Halter

April 2, 2014

michael.halter@nist.gov

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Summary

• The ability to generate measurements that 

characterize a wide-field fluorescence microscope 

is useful to ensure nominal instrument 

performance.

• The procedure described here benchmarks the 

microscope to a commercial fluorescent glass

that has similar excitation and emission 

properties to FITC, GFP, etc.

• The procedure can be expanded to benchmark 

other fluorescent channels when suitable 

reference materials are available.

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Limitations

• This is a beta-test

– Only tested with ~10 different widefield

microscope systems with CCD detectors (not 

CMOS) and 10x magnification objectives

– GG 475 Schott glass fluorescence is excited 

between 450 nm-470 nm (lower wavelengths 

can cause photobleaching) and emits 520 nm –

700 nm 

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Step 1: Prerequisites

• Manual or automated wide field fluorescence microscope 

with 10x objective ~0.15-0.3 NA1 and a CCD camera

• Filter set to image FITC or similar fluorophore.

• Ensure there is a Micro-Manager driver available for your 

microscope camera.

-http://www.micro-manager.org/wiki/Device%20Support

(tested with Roper CoolSnap FX, HQ, and HQ2; Zeiss AxioCam; ThorLabs

DCC1545, Retiga 2000R)

1May also work with other objectives. 4
Step 2: Things you need

• Download

– MicroManager (w/ImageJ) to the computer that will control the camera on 

the microscope (http://www.micro-manager.org/wiki/Download_Micro-Manager_Latest_Release)

– MicroManager benchmarking script 

(http://www.nist.gov/mml/bbd/cell_systems/upload/Microscope_Performance_v1.2.txt)

• Identify a computer with a printer and Excel to use for control charting of 

benchmarking metrics

– Download Excel spreadsheet template for control charting 

(http://www.nist.gov/mml/bbd/cell_systems/upload/Microscope_Charting Template_v2.xlsx)

• Schott Glass GG475 (25 mm dia x 3 mm thick)1

• Sample holder for the Schott Glass2

• Spatial calibration slide/stage micrometer

1Available from Edmund Scientific and other filter suppliers. Other fluorescent 

materials can be used for benchmarking. The material should be photostable

and homogenously fluorescent to facilitate accurate benchmarking.

2See next page.

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Example Sample Holders

Hole in polystyrene sample holders was cut with a Dremel tool. 

Rough edges were removed by scraping with a razor blade.

Schott Glass 

GG475 

6 well plate

Universal stage 

adapter

10 cm dish

35 mm dish

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Step 3: Install and Start MicroManager

(skip this step if you already run MicroManager)

• Turn on camera and microscope. Set microscope filters to FITC (or similar 

filter set) and place 10x objective under specimen location.

• Install MicroManager using instructions at the MicroManager website 

(http://www.micro-manager.org/wiki/Micro-Manager_User%27s_Guide#Installation).

• Start MicroManager.

• Configure MicroManager to control your microscope camera 

– If you have not previously installed MicroManager:

• Tools > Hardware Configuration Wizard > Create new configuration

• Select the camera and press Add

• Accept the remainder of settings using defaults values

• Place fluorescent glass on the microscope stage

• Test microscope operation by starting live acquisition and make sure you 

can capture fluorescent images of the Schott glass. TURN OFF LIVE 

ACQUISITION MODE

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Step 4: Install and Run Benchmarking Script 

• From MicroManager menu

– Tools > Scrip Panel > Open (Microscope_Performance_v1.1.bsh)

– Run

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Step 5a: Data Display and Spatial 

Calibration Window

• Click Data Display checkbox.

• If the spatial calibration is known, the value can be entered in 

text box. If not, check the box indicating that you will 

generate a calibration.

• Press OK.

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Step 5b. Spatial Calibration

• Place the spatial calibration slide on the stage and focus. 

Then, press OK.

• Draw a line between two calibration points then click OK.

• In the next dialog input the distance of the line in 

micrometers

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Step 6. Insert Fluorescent Glass Sample and Focus

• Place the sample holder and glass reference material 

on the sample stage. 

• Set the focus

• Click ‘OK’ on the open dialog box

1. Move the stage so that the edge 

of the fluorescent glass in in the 

field of view. 

2. Focus on the edge of the 

fluorescent materials proximal 

to the microscope objective.

3. Move the stage to a position 

where the field of view is filled 

with the fluorescent reference 

material.

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Example of edge in focus
Step 7: Setting up background 

fluorescence measurement

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• Carefully remove Schott glass from the sample 

holder and store in a safe place

• Make no other changes to the microscope system 

and click ‘Ok’ in the open dialog box
• Copy the data from the Benchmark Performance Parameters 

table of results

– Ctrl-A to select all 

– Ctrl-C to copy

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Step 8: Charting
Step 8: Charting

• Open Charting Template in Excel

– If using for the first time, delete example data from the Charting Template

• Paste the Benchmark Performance Parameters into the appropriate column

• You will need to remove the column with the parameter descriptions that is 

automatically copied from the MicroManager results table.

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Delete example data 

Record new Benchmark Performance 

Parameters in appropriate column. 

Data and statistics used for charting 

(DO NOT CHANGE).

Each Chart is automatically updated 

and stored as separate Excel Chart 

Sheet.

Add Date and User Name with 

each set of benchmark data.
Step 8: Charting

• Detection Threshold, Saturation, Intensity Calibration, and Mean Intensity at Saturation are 

critical metrics that should be charted to ensure nominal microscope operation. 

• Each plot should automatically update when new numbers are recorded.

• Save Excel file with charting data. Plots should be printed each time new data is entered for 

evaluating the microscope performance. 

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The plots should be stored in a notebook near the 

microscope or taped to the wall near the microscope 

to display the trend of the benchmarking metrics.
Brief Definitions of Charted 

Parameters

• Detection Threshold1: This is a measure of the sensitivity of your 

microscope to detecting low levels of light. It is the shortest possible exposure time 

when the SNR of fluorescence emitted from the glass is greater than or equal to 3.

• Saturation1: This is a measure of when your detector response becomes nonlinear. It is the exposure time at which the pixel variance plotted against intensity 

becomes non-linear.

• Intensity Calibration1: This can be used to calibrate for day-to-day 

variations on your microscope. It is the counts/(10 m2  s) detected from the 

fluorescent glass used as a reference. 

• Mean Intensity at Saturation1: This is a measure of when your 

detector response becomes non-linear. Similar to “Saturation”, but provided in 

units of “counts” on the detector. In general, intensity measurements should be 

below this value to remain in the linear range.

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1A more in-depth description of these parameters and how they are determined is available in the 

publication, “Performance Benchmarking and Intensity Calibration of a Widefield Fluorescence 

Microscope Using Fluorescent Glass” Cytometry A, in review (2014) 
Interpreting Changes in Charted 

Parameters

• Detection Threshold, Saturation, and Intensity Calibration 

all increase or decrease proportionally

– Causes: Illumination source fluctuation or a beam splitter or neutral 

density filter placed in light path

• Detection Threshold only changes

– Indicates a change in the noise floor of the detector. Could be from 

changing the read rate setting on detector, the temperature of the 

detector or degradation of the sensor components 

• Other combinations of changes

– The parameters will likely change when the optical components of the 

microscope or settings on the detector are changed, sometimes in nonobvious ways. Changes in the parameters in the absence of any hardware 

or software changes indicates drift in the measurement system. 

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