Time-gated flow cytometry: an ultra-high selectivity method to recover ultra-rare-event μ-targets in high-background biosamples

Jin, Dong-Yan; Piper, James A.; Leif, Robert C.; Yang, Sean; Ferrari, Belinda C.; Yuan, Jingli; Wang, Guilan; Vallarino, L. M.; Williams, John W

doi:10.1117/1.3103770

Cited by 35 publications

(26 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The averaged intensity of 69.1 V corresponds to signal-to-background ratio of 21.6. However, this CV value of 27% was larger than the original value of 7% as measured by the time-gated luminescent flow cytometry (3,22). We attribute the relatively large CV of the prototype to the position difference of targeted cells.…”

Section: Prototype Calibrationmentioning

confidence: 77%

“…These include both flow cytometry-based techniques and solid-phase image scanning cytometry techniques (3,4,(6)(7)(8). The latter usually uses a planar multielement photodetector, such as a charge-coupled device (CCD) camera, to make images from adjacent elements across the specimen, followed by scene segmentation and use of pattern recognition algorithms to locate the particular targets (9)(10)(11)(12)(13)(14)(15).…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Lu¹,

Jin²,

Leif³

et al. 2011

Cytometry Pt A

Self Cite

View full text Add to dashboard Cite

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

show abstract

Section: Prototype Calibrationmentioning

confidence: 77%

mentioning

confidence: 99%

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

Lu¹,

Jin²,

Leif³

et al. 2011

Cytometry Pt A

Self Cite

View full text Add to dashboard Cite

show abstract

“…Time-gated luminescence (TGL) technique is drawing increasingly more attentions in the analytical fields of microbiology and molecular biology, medical diagnostics, health and environmental researches as well as applications [1][2][3][4]. It takes advantage of long-lived luminescence from special substances, such as lanthanides exhibiting emission lifetime of >100 ȝs, which overwhelms autofluorescence, whose lifetime is usually < 0.1 ȝs.…”

Section: Introductionmentioning

confidence: 99%

Cytometric investigation of rare-events featuring time-gated detection and high-speed stage scanning

Piper

Huo

et al. 2011

2011 International Quantum Electronics Conference (IQEC) and Conference on Lasers and Electro-Optics (CLEO) Pacific Rim Incorpo

Self Cite

View full text Add to dashboard Cite

show abstract

“…A frequency-quadrupled Nd:YVO 4 (and Nd:YAG) laser at 266 nm can excite most lanthanide; however, such a deep UV is too destructive to the biology system and requires the optics to function at that short wavelength. Although recent solid-state laser research has demonstrated pulsed laser sources for this wavelength band (27,28), they are far more expensive compared with the light emitting diode (LED) sources (10,29). Another problem arising from this bandwidth is that the routine microscopes have too low transmission to deliver the power to the specimen, since most microscopy objectives and slide cover slips have the cuton edge from 330 to 340 nm.…”

mentioning

confidence: 99%

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

Jin

2011

Cytometry Pt A

Self Cite

View full text Add to dashboard Cite

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...

show abstract

Time-gated flow cytometry: an ultra-high selectivity method to recover ultra-rare-event μ-targets in high-background biosamples

Cited by 35 publications

References 40 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

Cytometric investigation of rare-events featuring time-gated detection and high-speed stage scanning

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

Contact Info

Product

Resources

About