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

Abstract

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...

How to Build a Time‐Gated Luminescence Microscope

“…At the end of this protocol, we describe steps to synchronize the pulsed excitation and gated detection. Refer to Jin (2011b) and Jin and Piper (2011).…”

“…The idea and prototype demonstration of optimizing a mechanical chopper suitable for time-gated detection were reported by the authors of this unit in 2011 (Jin, 2011b;Jin and Piper, 2011). The assembly of the time-gated luminescence chopper unit is displayed in Figure 2.22.3.…”

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

“…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.…”

Practical Implementation, Characterization and Applications of a Multi-Colour Time-Gated Luminescence Microscope

“…In our previous work, we demonstrated a low-cost true-colour TGL microscope, featuring an ultraviolet light-emitting diode (UV LED) for excitation and a mechanical chopper for time-gating 13 25 26 . In order to simultaneously excite multiple long-lived probes, especially terbium with sensitizing moiety complex that needs a triplet energy around 30,000 cm −1 to pump Tb 3+ to its excited state ( 5 D 4 ; 20,400 cm −1 ) 25 , efficient excitation at 300–340 nm is required; however, the power of currently available UV LEDs at this wavelength range is not strong enough.…”

“…In our previous work, we demonstrated a low-cost true-colour TGL microscope, featuring an ultraviolet light-emitting diode (UV LED) for excitation and a mechanical chopper for time-gating 13 25 26 . In order to simultaneously excite multiple long-lived probes, especially terbium with sensitizing moiety complex that needs a triplet energy around 30,000 cm −1 to pump Tb 3+ to its excited state ( 5 D 4 ; 20,400 cm −1 ) 25 , efficient excitation at 300–340 nm is required; however, the power of currently available UV LEDs at this wavelength range is not strong enough. On the other hand, flash lamps emitting deep UV with high power but low repetition rate (less than 100 Hz) have been used for TGL microscopy 27 28 , but the long detection windows prevent efficient collection of lanthanide luminescence typically with decay lifetimes of ~1 ms or less, as well as having stability issues when synchronized to mechanical choppers.…”