Russell Connally

Practical time‐gated luminescence flow cytometry. II: Experimental evaluation using UV LED excitation

In the previous article [Part 1 (8)], we have modelled alternative approaches to design of practical time‐gated luminescence (TGL) flow cytometry and examined the feasibility of employing a UV LED as the excitation source for the gated detection of europium dye labelled target in rapid flow stream. The continuous flow‐section approach is well suited for rare‐event cell counting in applications with a large number of nontarget autofluorescent particles. This article presents details of construction, operation and evaluation of a TGL flow cytometer using a UV LED excitation and a gated high‐gain channel photomultiplier tube (CPMT) for detection. The compact prototype TGL flow cytometer was constructed and optimised to operate at a TGL cycle rate of 6 kHz, with each cycle consisting of 100 μs LED pulsed excitation and ∼60 μs delay‐gated detection. The performance of the TGL flow cytometer was evaluated by enumerating 5.7 μm Eu3+ luminescence beads (having comparable intensity to europium‐chelate‐labeled Giardia cysts) in both autofluorescence‐rich environmental water concentrates and Sulforhodamine 101 (S101) solutions (broadband red fluorescence covering the spectral band of target signals), respectively.

Long-lived visible luminescence of UV LEDs and impact on LED excited time-resolved fluorescence applications

We report the results of a detailed study of the spectral and temporal properties of visible emission from three different GaN-based ultraviolet (UV) light emitting diodes (UV LEDs). The primary UV emission in the 360–380 nm band decays rapidly (less than 1 µs) following switch-off; however, visible luminescence (470–750 nm) with a decay lifetime of tens of microseconds was observed at approximately 10−4 of the UV intensity. For applications of UV LEDs in time-resolved fluorescence (TRF) employing lanthanide chelates, the visible luminescence from the LEDs competes with the target Eu3+ or Tb3+ fluorescence in both spectral and temporal domains. A UV band-pass filter (Schott UG11 glass) was therefore used to reduce the visible luminescence of the UV LEDs by three orders of magnitude relative to UV output to yield a practical excitation source for TRF.

High resolution detection of fluorescently labeled microorganisms in environmental samples using time‐resolved fluorescence microscopy

The high level of discrimination offered by fluorescence microscopy has led to its widespread use for the analysis of individual microbial cells. The major limitation of fluorescence microscopy in microbial ecology is that many types of environmental samples contain autofluorescent material that can obscure emission from a fluorescent label. Time-resolved fluorescence microscopy (TRFM) is a technique that greatly reduces background autofluorescence whilst maintaining signal strength of the fluorescent target. TRFM differs from fluorescent microscopy in the use of fluorophores that are characterized by long-lived luminescence. Samples are briefly illuminated to excite fluorescence then capture of luminescence is delayed for a time interval sufficient to ensure autofluorescence has largely faded. TRFM has not been extensively used in microbiology because of the limitations and cost of available time-resolved microscopes and the lack of suitable long-lived fluorescent labels. Here we describe modification of a commercial fluorescence microscope for time-resolved operation through the addition of an image-intensified camera and low cost flashlamp. The TRFM was used in combination with a novel immunofluorophore for the specific detection of Giardia cysts in a water sample containing large amounts of autofluorescent material. A 60-mus gate delay between excitation and detection resulted in a 30-fold increase in contrast of labeled parasites compared to conventional immunostaining. To our knowledge, this is the first report of the use of TRFM for the detection of microorganisms in environmental samples.

Time-gated luminescence microscopy

Autofluorescent algal samples were spiked with europium beads for analysis on a novel all‐solid‐state, time‐gated luminescence (TGL) microscope. Pulsed UV excitation (365 nm) was provided by a high‐power UV‐LED source fitted to an Olympus BX51 microscope. An “Impactron” electron multiplying charge‐coupled‐device (CCD) camera acquired images in delayed luminescence mode. Second, we evaluated sensitivity of the instrument by acquiring images of immunofluorescently labeled Giardia cysts with a single‐exposure period of 3 ms. The camera was triggered 3 μs after the LED had extinguished to yield a 14‐fold increase in signal‐to‐noise ratio within a single 33 ms capture cycle. This novel instrument could be switched instantly from prompt epifluorescence mode to TGL mode for suppression of short‐lived fluorescence.

High intensity solid-state UV source for time-gated luminescence microscopy

The unique discriminative ability of immunofluorescent probes can be severely compromised when probe emission competes against naturally occurring, intrinsically fluorescent substances (autofluorophores). Luminescence microscopes that operate in the time‐domain can selectively resolve probes with long fluorescence lifetimes (τ > 100 μs) against short‐lived fluorescence to deliver greatly improved signal‐to‐noise ratio (SNR). A novel time‐gated luminescence microscope design is reported that employs an ultraviolet (UV) light emitting diode (LED) to excite fluorescence from a europium chelate immunoconjugate with a long fluorescence lifetime.

Flash lamp-excited time-resolved fluorescence microscope suppresses autofluorescence in water concentrates to deliver an 11-fold increase in signal-to-noise ratio.

The ubiquity of naturally fluorescing components (autofluorophores) encountered in most biological samples hinders the detection and identification of labeled targets through fluorescence-based techniques. Time-resolved fluorescence (TRF) is a technique by which the effects of autofluorescence are reduced by using specific fluorescent labels with long fluorescence lifetimes (compared with autofluorophores) in conjunction with time-gated detection. A time-resolved fluorescence microscope (TRFM) is described that is based on a standard epifluorescence microscope modified by the addition of a pulsed excitation source and an image-intensified time-gateable CCD camera. The choice of pulsed excitation source for TRFM has a large impact on the price and performance of the instrument. A flash lamp with rapid discharge characteristics was selected for our instrument because of the high spectral energy in the UV region and short pulse length. However, the flash output decayed with an approximate lifetime of 18 micros and the TRFM required a long-lived lanthanide chelate label to ensure that probe fluorescence was visible after decay of the flash plasma. We synthesized a recently reported fluorescent chelate (BHHCT) and conjugated it to a monoclonal antibody directed against the waterborne parasite Giardia lamblia. For a 600-nm bandpass filter set and a gate delay of 60 micros, the TRFM provided an 11.3-fold improvement in the signal-to-noise ratio (S/N) of labeled Giardia over background. A smaller gain in an SNR of 9.69-fold was achieved with a 420-nm longpass filter set; however, the final contrast ratio between labeled cyst and background was higher (11.3 versus 8.5). Despite the decay characteristics of the light pulse, flash lamps have many practical advantages compared with optical chopper wheels and modulated lasers for applications in TRFM.

Practical Time-Gated Luminescence Flow Cytometry. I: Concepts

The method of time‐gated detection of long‐lifetime (1–2,000 μs) luminescence‐labeled microorganisms following rapid excitation pulses has proved highly efficient in suppressing nontarget autofluorescence (<0.1 μs), scatterings, and other prompt stray light (Hemmila and Mukkala, Crit Rev Clin Lab Sci 2001;38:441–519). The application of such techniques to flow cytometry is highly attractive but there are significant challenges in implementing pulsed operation mode to rapid continuous flowing sample to achieve high cell analysis rates (Leif R, Vallarino L, Rare‐earth chelates as fluorescent markers in cell separation and analysis, In: Cell Separation Science and Technology, ACS Symposium Series 464, American Chemical Society, 1991, pp 41–58; Condrau et al., Cytometry 1994;16:187–194; Condrau et al., Cytometry 1994;16:195–205; Shapiro HM, Improving signals from labels: Amplification and other techniques, In: Practical Flow Cytometry, 4th ed., Wiley, New York, 2002, p 345). We present here practical approaches for achieving high cell analysis rates at 100% detection efficiency, using time‐gated luminescence (TGL) flow cytometry. In particular, we report that new‐generation UV LEDs are practical sources in TGL flow cytometry.

Detection of Staphylococcus aureus with a fluorescence in situ hybridization that does not require lysostaphin.

To detect with whole‐cell fluorescence in situ hybridization (FISH), Staphylococcus aureus is typically permeabilized with lyozyme and lysostaphin. We tested whether it was feasible to detect S. aureus and differentiate it from Staphylococcus epidermidis with lysozyme‐only permeabilization. We compared lysozyme permeabilizationto S. aureus permeabilized with lysozyme in combination with lysostaphin. It was determined that S. aureus treated with agarose, methanol, and lysozyme could be detected with FISH. The 1 hr protocol is a useful alternative to conventional FISH. J. Clin. Lab. Anal. 25:142–147, 2011.

Dimethyl formamide-free, urea-NaCl fluorescence in situ hybridization assay for Staphylococcus aureus

Aims:  To test the feasibility of identifying Staphylococcus aureus with a fluorescence in situ hybridization (FISH) assay that uses a single hot‐plate and urea‐NaCl reagents.

Solid-state time-gated luminescence microscope with ultraviolet light-emitting diode excitation and electron-multiplying charge-coupled device detection

Many naturally occurring materials are autofluorescent, a property that can reduce the discriminative ability of fluorescence methods, sometimes to the point where they cannot be usefully applied. Shifting from the spectral to the temporal domain, it is possible to discriminate fluorophores on the basis of their fluorescence decay lifetime. Luminophores with sufficiently long lifetimes can be discriminated from short-lived autofluorescence using time-gated luminescence (TGL). This technique relies upon the application of a brief excitation pulse followed by a resolving period to permit short-lived autofluorescence to decay, after which detection is enabled to capture persistent emission. In our studies, a high-power UV LED was mounted in the filter capsule of an Olympus BX51 microscope to serve as the excitation source. The microscope was fitted with an Andor DV885 electron-multiplying CCD (EM-CCD) camera with the trigger input synchronized to UV LED operation. Giardia lamblia cysts labeled with the europium chelate BHHST were analyzed against an autofluorescent background with the TGL microscope. The EM-CCD camera captured useful TGL images in real time with a single exposure cycle. With 4x frame averaging, images acquired in TGL mode showed a 30-fold improvement in SNR compared with conventional fluorescence microscopy.