How to Build a Time‐Gated Luminescence Microscope

Dayong Jin1, Yiqing Lu1, Robert C. Leif2, Sean Yang2, Megha Rajendran3, Lawrence W. Miller3

1 Advanced Cytometry Laboratories, MQ BioFocus Research Centre & Photonics Research Centre, Macquarie University, New South Wales, 2 Newport Instruments, San Diego, California, 3 Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 2.22
DOI:  10.1002/0471142956.cy0222s67
Online Posting Date:  January, 2014
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The sensitivity of filter‐based fluorescence microscopy techniques is limited by autofluorescence background. Time‐gated detection is a practical way to suppress autofluorescence, enabling higher contrast and improved sensitivity. In the past few years, three groups of authors have demonstrated independent approaches to build robust versions of time‐gated luminescence microscopes. Three detailed, step‐by‐step protocols are provided here for modifying standard fluorescent microscopes to permit imaging time‐gated luminescence. Curr. Protoc. Cytom. 67:2.22.1‐2.22.36. © 2014 by John Wiley & Sons, Inc.

Keywords: lanthanide; time‐gated luminescence; microscopy; autofluorescence

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Table of Contents

  • Introduction
  • Basic Protocol 1: Construction and Use of a Mechanical Chopper TgL Unit
  • Basic Protocol 2: Construction and Use of Camera with Analog Signal Summation
  • Support Protocol 1: Bead Sample Preparation
  • Basic Protocol 3: Preparation and Use of Time‐Gatable Intensified CCD Camera
  • Support Protocol 2: Preparation of Water/Oil Emulsion Standards for Microscope Calibration and Testing
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Construction and Use of a Mechanical Chopper TgL Unit

  • The basic components for modification of a filter cube for the pulsed excitation design used in this protocol are shown in Figure :
    • A high‐power UV LED, such as #NCCU033A, commercially available from Nichia Corp. at a cost of less than $100 (
    • A UV excitation filter, such as a colored glass band‐pass filter U330; 0.5‐in. diameter) available from Edmund Optics (
    • A standard dichroic filter, such as 400dclp filter, >400‐nm pass, from ( within a fluorescence microscopy filter cube, such as a standard Olympus fluorescence filter cube used in this work
    • An aspherical lens, such as #C110TME from Thorlabs (
    • This modification only requires one customized mechanical lens holder, as shown in Figure C. The holder can be machined from plastic or aluminum (in this work at cost of ∼ $200) with internal screws for fine tuning the aspherical lens position.
  • The basic components for construction of a gated mechanical chopper unit are shown in Figure :
    • An enclosed version of high‐speed chopper (C995 Optical Chopper) commercially available from, Terahertz Technologies ( at a cost of ∼$1000
    • A condenser lens (30 mm EFL, shown in Fig. B) available from Edmund Optics (
    • A standard eyepiece (×10) from commercial microscopes, such as Olympus microscopes
    • Other mechanical holders (shown in Fig. ), customized by the Advanced Cytometry Labs at Macquarie University, are necessary to minimize the chopper vibration and maximize the optical alignment accuracy. These aluminum mechanical holders and adaptors are usually machined under a precisely programmed cutting machine, with cost at ∼$4000 each job.
    • Stanford Research Signal Generator DG535, or other low‐cost signal generator

Basic Protocol 2: Construction and Use of Camera with Analog Signal Summation

  • Europium FireRed 5‐ and 0.5‐µm microspheres (Newport Instruments, part nos. EuFR5UM and EuFR0_5UM, respectively;
  • DNA‐Check beads, 10 µm diameter (Beckman Coulter, part no. 6603488,
  • The Nichia UV LED is the same as that described in protocol 1. As is shown in Figure A, a quartz condenser can be used to focus an LED that is substituted for the excitation lamp of a Leica fluorescent microscope. The UV LED is positioned close to the back of a LINOS condenser, which is attached to the excitation entrance of a fluorescence microscope (Leif et al., ). The mounting system described below can be used to mount any small light weight object that needs to be centered including a LED or an optical fiber. Nichia can supply a heat sink attached to the LED for free.
  • A Leitz MPV II fluorescence epi‐illuminated microscope or equivalent equipped with a 10× 0.25 NA, a 40× 0.65 NA, and an infinity‐corrected objective high‐ultraviolet‐transmission UPL Fluorite 60 oil, NA 1.25, with aperture (Olympus part no. 1UB532), was employed to observe and to electronically photograph the beads
  • The UV fluorescence was excited at 365 nm with a similar UV LED from the same manufacturer, Nichia, that was employed protocol 1
  • The emitted light was observed through an Omega Optical ( PloemoPak cube UV DAPI, equipped with a 365‐nm narrow‐band‐width excitation filter (Omega 365HT25) and a 400‐nm beam‐splitter (Omega 400DCLP02). The CCD optical path was optionally equipped with either a 619 nm narrow‐band emission filter (Omega 618.6NB5.6) or a standard DAPI 450 nm emission filter (Omega 450DF65).
  • Qioptiq LINOS (http://www.qioptiq‐ Combi. Condenser (16/21.4 mm; part G063011000)
  • The parts for construction of the Condenser Mount and LED Mount were available at the precision instrument shop and the local hardware store
  • The basic components are shown in Figures and . The Laserlab supply was used to power the UV LED in pulsed and continuous mode. 1‐msec wide pulses were delivered at up to 500 Hz. The images were triggered by the trailing edge of the pulses and had an exposure time of 1 msec or longer, as specified. The square‐wave pulse train was provided by the Global Specialties Instruments signal generator. The Leitz fluorescent microscope is equipped with 365‐nm pulsed excitation from a Nichia 230mW LED (Leif et al., ; Jin et al., ). The Atik 314L+ camera was cooled (ΔT = −27°C).
  • Laserlab LED power supply (; the custom‐built unit specified in protocol 1, or any other that will follow a function generator with at least a frequency range of 0 to 100 kHz and provide the peak power suggested by the manufacturer, should suffice.
  • Signal generator (Global Specialties Instruments model 4001; this unit is inexpensive but still adequate. One other alternative is a board in a PC that can serve as a function generator and possibly an oscilloscope. The caveat, as always, is to make sure that the software is both adequate and easy to use.
  • Oscilloscope: any dual trace should be suitable or as stated above a PC board should be considered
  • Camera: Artemis Atik 314L+ (‐ccd‐fs‐range.html). The Atik 314L+ can be cooled (ΔT = −27°C} and employs a 1392 × 1040–pixel Sony ICX‐285AL chip (pixel size of 6.45 × 6.45 µm2). This camera has a readout noise of 4 electrons and is equipped with a 16‐bit ADC and USB 2.0 interface. It should be purchased with a C‐mount adapter or as needed to fit the emission filter assembly. The Atik 314L+ camera has been succeeded by the FS14.
  • Emission filter wheel unit. The use of a commercial one is recommended, such as the Atik Electronic Filter Wheel 2, which is software and mechanically compatible with the. Atik 314L and its successor.
  • Artemis Capture software (

Support Protocol 1: Bead Sample Preparation

  • DNA‐Check beads (Beckman Coulter, part no. 6603488)
  • FireRed 5‐ and 0.5‐µm beads that contain Eu(TTFA) 3 (Newport Instruments;
  • HERMLE Z‐180‐M microcentrifuge (http://www.hermle‐
  • Branson Ultrasonifier model 450 ( with micro‐tip
  • Centrifugal Cytology Buckets (Newport Instruments)
  • Plain frosted end glass slide (Fisher Scientific, cat. no. 12‐550‐15)
  • Beckman GPR centrifuge (
  • Glass coverslips

Basic Protocol 3: Preparation and Use of Time‐Gatable Intensified CCD Camera

  • The required components and their relevant capabilities for ICCD‐based, time‐gated luminescence imaging are listed in Table 2.22.2. The system reported by Miller and Gahlaut (2010) that serves as the example here was built around a Zeiss Axiovert 200 microscope, but any inverted or upright wide‐field microscope will suffice (Gahlaut and Miller, ). As excitation illumination is delivered through the objective, a lens with adequate UV transmission is necessary. Here, a 63× objective with ∼50% transmittance at 350 nm was used (EC Plan Neofluar, 63× 1.25 N.A., Carl Zeiss). A collimated, UV LED (e.g., Mic‐LED‐365, Prizmatix, Ltd., emitting at 365 nm is required for excitation. The LED must be capable of external, TTL‐mediated switching with µsec‐scale rise/fall times, must have adjustable output (up to ∼50 mW at the exit window), and must be fittable to the epi‐illumination port of the microscope. The Prizmatix LED is one of several commercially available units that meet these requirements. A pulse generator with at least two output channels (TTL) and programmable burst mode operation (e.g., DG645, Stanford Research Systems) is needed to switch the LED and synchronize its operation with the intensifier component of the camera.
  • With respect to the type of intensified CCD camera, there is some latitude in selecting components. Firstly, it is possible to separately purchase an intensifier and lens‐couple it to a CCD. This option is more cost effective if one already has a suitable CCD camera; however, lower resolution, lower coupling efficiency, and greater complexity in hardware integration may be expected in comparison to a fiber‐optically coupled, integrated ICCD platform. Whether lens coupling or fiber coupling is chosen, the type of photocathode incorporated into the intensifier has the greatest impact on system performance. GenIII photocathodes have peak quantum efficiencies of 40% to 50% with sensitivity ranging from near‐UV to IR, depending on the particular material (GaAs or GaAsP; with or without ion barrier film). Miller employed an integrated ICCD system (Mega‐10EX, Stanford Photonics) that consists of a filmless, GaAsP photocathode (quantum efficiency = ∼0.4 from 450 to 600 nm) fiber optically coupled to a Sony XX285 CCD sensor. The CCD sensor is a 1380 × 1024 array of 6.47 µm pixels, and the effective pixel size of the intensifier/camera is 10.35 µm. The CCD can be read out at a full‐frame rate of 15 frames‐per‐sec.
  • This protocol assumes computer control of the ICCD system, and proprietary software packages are available from most camera manufacturers (e.g., Piper, v2.4.05, Stanford Photonics). At minimum, a computer and software are required that allow the user to control CCD frame length, and that can generate an external TTL trigger pulse synchronous with the beginning of image acquisition. Additionally, the camera system must allow for external TTL gating of the intensifier and on‐chip integration.
Table 2.2.2   MaterialsNecessary Hardware Components and Relevant Specifications for Building a Time‐Gated Microscope Using an Intensified CCD Camera with Pulsed UV LED Excitation

Component Manufacturer/model no. a Specifications
Epi‐fluorescence microscope Carl Zeiss, Inc. Axiovert 200 High N.A. (≥1.0) objective lens with ≥50% transmittance at 350 nm (e.g., EC Plan Neofluar, 63×, 1.25 NA)
Collimated UV LED (365 nm emission) Prizmatix, Ltd. MIC‐LED‐365 External on/off modulation (TTL) with ∼1 µsec rise/fall time
Adjustable output to at least ∼50 mW at exit window
Digital pulse generator Stanford Research Systems, Inc. DG645 At least 2 independent delay outputsProgrammable burst mode capabilityExternally triggered start (TTL)
Intensified CCD camera Stanford Photonics, Inc. MEGA‐10EX GenIII photocathode, peak Q.E. = ∼0.4External triggering (TTL) of intensifierOn‐chip integrationAdjustable intensifier gain voltage

 aComponents listed here are for system originally described in (Gahlaut and Miller, ).

Support Protocol 2: Preparation of Water/Oil Emulsion Standards for Microscope Calibration and Testing

  • Tris base
  • NaCl
  • 1 M HCl
  • Mineral oil (Sigma‐Aldrich, cat. no. M5904)
  • Span 80 (Sigma‐Aldrich, cat. no. 85548)
  • Tween 80 (Sigma‐Aldrich, cat. no. P1754)
  • Lumi4 (Lumiphore, Inc.,
  • TbCl 3·6H 2O (Sigma‐Aldrich, cat. no. 204560)
  • Valap (1:1:1 mixture of vaseline, lanolin, and paraffin)
  • Volumetric glassware (for buffer preparation)
  • pH meter (for buffer preparation)
  • Separatory funnel, 60 ml (e.g., Chemglass, cat no. CG‐1743‐07)
  • Glass reaction/storage vials, 20 ml (e.g., Chemglass, cat no. CG‐4904‐03)
  • Magnetic stir bar, 7 mm × 2 mm (e.g., Chemglass, cat no. CG‐2003‐14)
  • Bench‐top magnetic stirrer
  • Double‐sided adhesive tape
  • Microscope slides
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