Time-Gated Luminescence Microscopy Allowing Direct Visual Inspection of Lanthanide-Stained Microorganisms in Background-Free Condition

Jin, Dayong; Piper, James A.

doi:10.1021/ac103207r

Cited by 124 publications

(114 citation statements)

References 49 publications

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“…After an eyepiece (103, Stock No. NT36-130, Edmund Optics) at the detection port, a compact mechanical chopper (C-995, Terahertz Technologies) was positioned at the beam focus to generate a rapid on-off switching time (19,20). A second identical eyepiece was used to collect the diverging TGL into the detector, either a channel PMT (CPMT, MH1372, PerkinElmer) for rapid scanning of ''elements of interest" or a color CCD camera (2 mega pixels, DS-Vi1, Nikon) or the naked eye for visual confirmation.…”

Section: Methodsmentioning

confidence: 99%

Automated detection of rare‐event pathogens through time‐gated luminescence scanning microscopy

Lu¹,

Jin²,

Leif³

et al. 2011

Cytometry Pt A

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Many microorganisms have a very low threshold (\10 cells) to trigger infectious diseases, and, in these cases, it is important to determine the absolute cell count in a lowcost and speedy fashion. Fluorescent microscopy is a routine method; however, one fundamental problem has been associated with the existence in the sample of large numbers of nontarget particles, which are naturally autofluorescent, thereby obscuring the visibility of target organisms. This severely affects both direct visual inspection and the automated microscopy based on computer pattern recognition. We report a novel strategy of time-gated luminescent scanning for accurate counting of rare-event cells, which exploits the large difference in luminescence lifetimes between the lanthanide biolabels, [100 ls, and the autofluorescence backgrounds, \0.1 ls, to render background autofluorescence invisible to the detector. Rather than having to resort to sophisticated imaging analysis, the background-free feature allows a single-element photomultiplier to locate rare-event cells, so that requirements for data storage and analysis are minimized to the level of image confirmation only at the final step. We have evaluated this concept in a prototype instrument using a 2D scanning stage and applied it to rare-event Giardia detection labeled by a europium complex. For a slide area of 225 mm 2 , the time-gated scanning method easily reduced the original 40,000 adjacent elements (0.075 mm 3 0.075 mm) down to a few ''elements of interest'' containing the Giardia cysts. We achieved an averaged signal-to-background ratio of 41.2 (minimum ratio of 12.1). Such high contrasts ensured the accurate mapping of all the potential Giardia cysts free of false positives or negatives. This was confirmed by the automatic retrieving and time-gated luminescence bioimaging of these Giardia cysts. Such automated microscopy based on time-gated scanning can provide novel solutions for quantitative diagnostics in advanced biological, environmental, and medical sciences. ' 2011 International Society for Advancement of Cytometry

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Section: Methodsmentioning

confidence: 99%

Automated detection of rare‐event pathogens through time‐gated luminescence scanning microscopy

Lu¹,

Jin²,

Leif³

et al. 2011

Cytometry Pt A

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“…The light emitted at a large solid angle ([458) is collected by a UV-fused silica plano-convex lens (f 5 35.mm; Ø51 inch, Thorlabs LA4052UV), which is then redirected by a dichroic mirror (365 DCLP BS, http://www.chroma.com/) into the UV objective. Though mechanical chopper gating has been reported with slow switching times of 100-200 ls (14,32-36), we employed a new compact fast chopper (C995 Optical Chopper, Terahertz Technologies, NY; 167 rotating cycles per second) (30,37) and a new optics configuration to achieve up to 11 ls switching time at 2.5-kHz repetition rate with a transparent/blocking duty ratio of 75%:25% (30). This duty ratio is achieved by laser micromachining of every second blades from original 30 blades down to 15 blades.…”

Section: Methodsmentioning

confidence: 99%

“…The LED pulses were synchronized to a pinhole-assisted optical chopper unit allowing naked eye observation of time-gated bioimages (30). A commercially available relatively inexpensive UV objective (403, 240-360 nm, NA 5 0.50, a OFR objective from Thorlabs, Newton, NJ, USA) was employed for efficient UV delivery in a standard epifluorescence microscopy structure.…”

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confidence: 99%

Demonstration of true‐color high‐contrast microorganism imaging for terbium bioprobes

Jin

2011

Cytometry Pt A

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Lanthanide bioprobes offer a number of novel advantages for advanced cytometry, including the microsecond luminescence lifetime, sharp spectral emission, and large stokes shift. However, to date, only the europium-based bioprobes have been broadly studied for time-gated luminescence cell imaging, though a wide range of efficient terbium bioprobes have been synthesized and some of them are commercially available. We analyze that the bottleneck problem was due to the lack of an efficient microscope with pulsed excitation at wavelengths of 300-330 nm. We investigate a recently available 315 nm ultraviolet (UV) light emitting diode to excite an epifluorescence microscope. Substituting a commercial UV objective (403), the 315 nm light efficiently delivered the excitation light onto the uncovered specimen. A novel pinhole-assisted optical chopper unit was attached behind the eyepiece for direct lifetime-gating to permit visual inspection of background-free images. We demonstrate the use of a commercial terbium complex for high-contrast imaging of an environmental pathogenic microorganism, Cryptosporidium parvum. As a result of effective autofluorescence suppression by a factor of 61.85 in the time domain, we achieved an enhanced signal-to-background ratio of 14.43. This type of time-gating optics is easily adaptable to the use of routine epifluorescence microscopes, which provides an opportunity for high-contrast imaging using multiplexed lanthanide bioprobes. ' International Society for Advancement of CytometryKey terms time-gated luminescence; bioimaging; terbium; UV LED; Cryptosporidium; high-contrast EPIFLUORESCENCE microscopes are routinely found in both research and diagnostic laboratories. From the practical point of view, cytometrists expect a microscope to be low cost, compact, easy-to-operate at high sensitivity, including the capacity to discriminate against nontarget substance, detect multiple species at once, and have high throughput. Time-domain discrimination of targeted microorganisms using long-lifetime lanthanide bioprobes shows promise in meeting these demands. This is due to the ease of time-gating of probes that have lifetimes of several hundreds of microseconds, which eliminates the autofluorescent background of several nanoseconds (1). In practice, however, there are several bottleneck problems challenging both the instrumentation and bioprobe developments. To date, only the europiumbased bioprobes (excited at 330-420 nm, emitting 615 nm at $10 nm bandwidth) have been broadly studied for time-gated luminescence cell imaging (1-15). This is due to the fact that other rare-earth ion complexes are either less efficient (16) [only europium and terbium complexes are less sensitive to vibrational quenching by energy transfer to OH, NH, or CH oscillators (15)] or require pulsed ultraviolet (UV) excitation below 330 nm (17). Another reason is the lack of near-infrared detection cameras. In fact, some cameras have included near-infrared blocking filters. For example, many efficient terbium-base...

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“…For instance, a very long delay time cuts out the most intense emission from the phosphorescent probe, and a very long acquisition time leads to noise integration at the end of the decay. If the luminescent probe decay is a single-exponential function, then the phosphorescence signal intensity decreases as in the range 0.5-2.5 ms so that, ideally, t 1 should be in the range 50-250 ms and t 2 in the range 1.5-7.5 ms. Because most of the TRL microscopes used to date are modifications of epifluorescence microscopes and are fitted with a combination of a fast switching pulsed light-emitting diode and a chopper [36], long acquisition times can be implemented. Reported windows are, however, in the range 0.1-5 ms only, which explains why actual improvements are less than predicted.…”

Section: Time-resolved Luminescence Microscopymentioning

confidence: 99%

Lighting up cells with lanthanide self-assembled helicates

Bünzli

2013

Interface Focus.

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Lanthanide bioprobes and bioconjugates are ideal luminescent stains in view of their low propensity to photobleaching, sharp emission lines and long excited state lifetimes permitting time-resolved detection for enhanced sensitivity. We show here how the interplay between physical, chemical and biochemical properties allied to microfluidics engineering leads to self-assembled dinuclear lanthanide luminescent probes illuminating live cells and selectively detecting biomarkers expressed by cancerous human breast cells.

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Time-Gated Luminescence Microscopy Allowing Direct Visual Inspection of Lanthanide-Stained Microorganisms in Background-Free Condition

Cited by 124 publications

References 49 publications

Automated detection of rare‐event pathogens through time‐gated luminescence scanning microscopy

Automated detection of rare‐event pathogens through time‐gated luminescence scanning microscopy

Demonstration of true‐color high‐contrast microorganism imaging for terbium bioprobes

Lighting up cells with lanthanide self-assembled helicates

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