Light Microscopy and Fluorescence Technology

The "Light microscopy and fluorescence technology" group is subdivided into tow groups namely:  
  1. Microscopy, software and instrumentation (Lennard Voortman)
  2. Development of point-of-care (POC) lateral flow assays using upconvertion phosphors as reporters (Paul Corstjens)

Microscopy, software and instrumentation (Lennard Voortman)

The group maintains and supports a modern facility for light microscopic imaging which relates to development of dedicated hard- and software for analysis of microscopic imaging, with special emphasis on the analysis of living cells. The instrumentation of the facility which is used by many departments of the LUMC consist of:

  • Three confocal laser scanning microscope systems (CSLM) (two of which equipped for two photon excitation) (Leica SP8, Leica SP5 and Leica SP5-STED);
  • Two automated system for time lapse recording of (GFP labeled) living cells (AF 6000);
  • A Fluorescence Lifetime Imaging Microscope (FLIM) to study molecular interactions in living cells on the basis of FRET;
  • Two workstations for the analysis of COBRA stained chromosomes;
  • Two digital fluorescence microscopes for quantitative cell analysis;

  Principal of FRAP

Principal of FLIM

Specific projects involve the construction of a new FLIM system, software for automated tracking of living cells, fast algorithms for real time deconvolution, methods to quantify molecular dynamics using FRAP and FLIP, or to track and analyze subcellular structures (e.g. telomeres).

Development of point-of-care (POC) lateral flow assays using upconvertion phosphors as reporters (Paul Corstjens)

Collaborators: Claudia de Dood, Elisa Tjon Kon Fat, Hans Tanke, Paul Corstjens 

General

Point-of-care (POC) testing is a cost effective alternative to many diagnostic tests. Successful POC testing largely depends on  the assay sensitivity that can be realised with relatively low cost reagents and equipment; especially, in situation where POC is applied in resource-poor settings. For this purpose we have employed a unique reporter, upconverting phosphor (UCP) particles, in combination with lateral flow (LF) assays. UCPs are submicron particles doped with lanthanides that convert infrared to visible light (a process called upconversion). In collaboration with industry (Qiagen Lake Constance) and academic partners (University of Pennsylvania; Dr. Haim Bau; New York University School of Dentistry: Dr. D. Malamud)) we have developed and applied low cost handheld readers and microfluidic cassettes for various applications in infectious diseases.

Upconverting phosphor technology

Upconverting phosphors (UCP) are submicron particles of ceramic materials doped with rare earth ions (lanthanides). Lanthanide ions offer the possibility to convert infrared radiation to visible light by upconversion. For instance, a two-step energy transfer process from Yb3+ to Er3+ converts IR photons of 980 nm (absorbed by Yb3+) to green photons (550 nm, emitted by Er3+). The efficiency of this process is high (up to 25%). The colour of the emitted light can be determined by the specific second lanthanide ion (e.g. Er3+: green, Tm3+: blue). The diameter of UCP particles used depends on the application and assay format from as small as 6 nm to as large as 400 nm. The unique anti-Stokes emission of UCPs and the fact that upconversion does not occur in biological samples or materials used for detection, enables autofluorescence-free detection of luminescence, which is key to the development of ultra-sensitive diagnostic assays. Moreover, UCPs are highly photostable and have narrow emissions that enable almost unlimited observation times and multiplexed detection. Nano UCPs can be prepared using a variety of methods such as solid state reaction, sol-gel precipitation methods, pool combustion, flame synthesis and in solution thermo hydrolysis. UCPs are usually silica-coated to facilitate chemical modification and subsequent conjugation to biomolecules, but also to prevent non-specific binding.

Up-converting Phospor Technology

Point-of-care (POC) testing

There is on-going development of POC tests as alternatives to diagnostic assays performed in specialized laboratories with sophisticated equipment. This trend is driven by advances in molecular biology and biochemistry and by the availability of disposable sample handling devices and cartridges for analysis. Also, handheld analysers are available for relatively low prices. POC testing in most cases implies a rapid test procedure.  Challenge of many POC tests is to provide sufficient sensitivity with both low-cost assays and equipment. UCPs are well suited as reporters for POC tests, e.g. LF tests and modern miniaturised desktop analysers, as they have high specific activity and the required detection instruments are simple and inexpensive.

Lateral flow assay

A lateral flow assay is based on the capture of a biomolecule from a clinical sample by an antibody pair specific for a unique target. In LF assays the sandwich is formed at a defined location on a LF strip (nitrocellulose or similar porous surface) where the first antibody is immobilized (the Test line, T). The second antibody is bound to the UCP particle. LF assays are easy to perform, deliver results fast and are low demanding with respect to required equipment. The simplest UCP-LF assays requires addition of the sample to a tube with dry UCP-reporter reagent, immediately followed by the addition of a LF strip in the tube.

We have developed two types of UCP-LF based assays:  Design #1 is a single flow format (SF), but may include a pre-flow solution phase; Design #2 is a consecutive flow format (CF) that uses three sequential flow steps that allow separation/partitioning of the sample flow and the reporter flow. The UCP-LF assay read outs are often presented as a Ratio, the signal measured at the Test (T) line divided by the signal measured at the Flow Control (FC) line. The FC line ensures complete flow through the material. A second possibility is to normalize the signal measured at T and FC lines to an Index (I) line, where the I line is a fixed amount of UCP particles immobilized on the LF strip or assay case. The I line is also serves to correct for variation in reader efficiencies when comparing results obtained by different LF strip scanners. Fig. 1 shows a schematic of a LF strip and the Ratio calculations that can be used to deal with batch to batch variation and differences between readers for proper inter-assay comparison. 


Figure 1. LF strip analysis – calculation of the Ratio for inter-assay comparison.

Signals measured at the Test (T) and Flow-Control (FC) line expressed as relative fluorescence units (RFUs) are device dependent. The Ratio (R) of the T and FC signals is device independent. The index (I) line contains a constant amount of UCP particles, that serves as a quality control line for reader efficiency; T and FC values can be normalized to the highest I value measured (Imax ) for a series of lateral flow strips. Background (zero) correction is applied only to the T line.

Single flow format

In the UCP-LF single flow (UCP-SF) assay format we typically use (dry) UCP reagents either added to the sample pad or integrated in a conjugate release pad. It is also possible to mix the sample with the reporter conjugate before adding the mixture to the LF strip (Fig. 2). A pre-flow incubation of sample and reporter conjugate improves the efficiency/yield of bound target antigen to the UCP reporter, which results in ~20-fold  increased analytical sensitivity compared to a fully optimized ELISA. The solution phase incubation is used for assays that require maximum sensitivity and highest reproducibility. We demonstrated that under optimal conditions detection in the order of  105-106 target molecules is feasible when using 4 mm width LF strips with 400 nm diameter Y2O2S:Yb3+,Tm3+ UCP reporter particles24,25. Hypothetically, this lower limit of detection implies that 0.01-0.1 pg of the 50 kDa molecule can be detected using UCP-LF. Assuming a sample input of 10 mL, the lowest detectable concentration would then be ~1-10 pg/mL. 



Figure 2. Single flow assay format (UCP-SF) for quantitative detection of amplified nucleic acids and soluble antigens.  

A: Sample preparation for nucleic acid detection, e.g. a 155 base pair HIV gag fragment; resulting amplicons incorporate digoxigenin (DIG) and biotin (BIO) hapten. B: Sample preparation for soluble antigen detection, e.g. extraction of HIV core protein p24. Solution phase: HIV amplicons bind via the DIG hapten to UCP coated with mouse anti-DIG antibodies (aDIG) and form UCP-DNA complexes; p24 molecules bind to UCP coated with mouse anti-p24 antibody (a1p24) and form UCP-p24 complexes. Lateral flow: UCP-DNA binds to the avidin via the BIO hapten on the DNA amplicon and UCP-p24 binds to a second a2p24. UCP particles that flow past the T line are capture at the FC line via anti-mouse antibodies (a Mouse).

This test format is also suitable to detect nucleic acids. We developed a rapid HIV detection assay including RNA purification and a short RT-PCR (25 cycles) utilizing UCP-SF (Fig. 3) with a large dynamic range and a sensitivity down to 300 virions per mL in serum (HIV-1 subtype B), which would be sufficient to detect HIV RNA in blood from newly infected as well as sero-converted individuals (after sero-conversion viral load drops but generally remains at a level of at several thousand).

Figure 3. RT-PCR amplification of a dilution series of  HIV-1(subtype B) analysed with UCP-SF.

A dilution series of a HIV-1 subtype B culture (300 through 108 virions/mL in normal human serum) was tested according the protocol illustrated in Fig. 2. Viral RNA was isolated and purified from 0.1 mL of sample using the ZR Viral RNA Kit (Zymo Research) and amplified using the Transcriptor One-Step RT-PCR Kit (Roche). DNA was eluted in 25 mL water and 1-10 mL was analysed with the UCP-SF assay. The lowest concentration tested (300 virions/mL) was detected using 10 m L eluted DNA.

Consecutive flow format


The UCP-LF consecutive flow (UCP-CF) was developed to detect multiple targets on a single LF strip with a generic UCP reporter, e.g. UCP particles coated with protein-A to identify immunoglobulins (Ig’s). In this generic type of assay the capture molecule on the LF Test line determines the assay specificity. Sample and UCP reporter are flowed separately in order to prevent saturation of the generic UCP reporter with non-target molecules. The sample is applied to the lateral flow strip first and chased onto the strip by wash buffer. The generic UCP reporter is then added to the sample pad in buffer and binds to the targets and the capture line as it flows (Fig. 4A).

Figure 4. Consecutive flow assay format (UCP-CF) for antibody detection.  

Panel A: Schematic for the detection of human anti-HIV antibodies using three sequential flow steps. Diluted serum sample is added to the LF strip allowing anti-HIV antibodies present in the serum sample to bind HIV-specific antigens on the T-line. The UCP reporter is coated with protein-A which binds to human IgG’s. The FC-line contains anti-IgG antibodies and will capture UCP particles that are not retained at the T-line. Panel B: Analysis of 100 serum samples from 49 sero-negatives and 51 sero-positives as determined with the OraQuick® HIV-1 Rapid Antibody test as gold standard. The highest negative value was 0.061.

 

POC test of infectious disease

The tests described below are developed with LUMC colleagues from the Department of Parasitology and the Department of Infectious Diseases

Most assays are focussed on applications for infectious diseases (Tab. 1) and their potential for multiplexing. For instance, an assay was developed that combined UCP-SF and UCP-CF formats for simultaneous antibody and cytokine detection for leprosy related antibody-mediated and cell-mediated immunity  responses. For several of these applications both low-tech as well as high-tech types of microfluidic devices were explored with the objective to integrate multiplexing  of different types of biomolecules in a single clinical sample.

Table 1. Examples of UCP-LF infectious disease applications (UCP-SF or UCP-CF format)

Pathogen

Pathogen Name

Target

Validated a

Viral

Hepatitus C Virus (HCV)

Antibody

Serum

Human Immunodeficiency Virus (HIV)

Core Protein (p24)

Recombinant protein

RNA (gag)

Serum

Antibody

Serum, Saliva

Human Papilloma Virus (HPV)

DNA

Carcinoma tissue

Influenza A Virus (H1N1)

DNA (unpublished)

Spiked saliva

Respiratory Syncytical Virus (RSV)

Antigens

Nasal wash

Bacterial

Bacillus cereus

DNA

Spiked saliva

Clostridium botulinum

Neurotoxin (BoNT/D)

Culture supernatant

Mycobacterium leprae

Cytokines (IFN-g, IL-10)

Serum, PBMCb

Antibody (aPGL-1)

Serum

Mycobacterium tuberculosis

Cytokines (IP-10, IL1-ß)

Whole bloodc

Chemokine (CCL4)

Whole bloodc

Antibody

Serum

Staphyloccus aureus

DNA

Purified DNA

Streptococcus pneumonia

DNA (lytA)

Purified DNA

Streptococcus pyogenes

DNA

Spiked saliva

Vibrio cholera

DNA (ctx)

Purified DNA

Plasmodium falciparum

DNA

Dried blood spots

Helminths

 

Taenia solium

Antibody

Serum

Schistosoma species

Glycoproteins (CAA CCA)

Serum, Urine

Antibody

Serum, Urine

 

a Refers to the type of sample used to evaluate the assay

b PBMC; peripheral blood mononuclear cells

c Whole blood samples; supernatant of heparinized blood after incubation of with (pathogen-specific) antigens

 

The UCP-LF antigen assays (UCP-SF) for detection of Schistosoma circulating antigens and TB/leprosy related cyto-/chemokine testing have evolved and are now available in a dry reagent format that allows convenient worldwide shipping. This dry-assay format is currently being evaluated by local staff at several institutions (hospitals and research laboratories) in 10 countries, mainly in Africa using UCP-LF strip readers such as the lightweight custom adapted ESEQuant LFR (referred to as UCP Quant; Qiagen Lake Constance GmbH, Stockach, Germany

The UCP-CF format assay has been mostly used for the (quantitative) detection of antibodies. The feasibility of dry reagents based on the utilization of UCP lyospheres (freeze dried pellets containing 100 ng of 400 nm Y2O2S:Yb3+,Tm3+ UCP particles; OraSure Technologies Inc., Bethlehem, PA, USA) was successfully investigated. For antibody detection UCP particles coated (by covalent coupling) with protein-A were employed. The UCP-CF format was shown to work equally well with newly available 40 nm diameter NaYF4:Yb3+,Er3+ materials4 (Intelligent Material Solutions Inc., Princeton, NJ, USA). As a result, the current focus is on the development of a lower cost dry reagent format analogous to the dry reagents used in the UCP-LF antigen assays (UCP-SF) but utilizing custom produced NaYF4-based nano materials. The UCP-CF assay was further demonstrated not only suitable for blood-based testing, but showed good performance in saliva and urine as well.

Microfluidics

Lateral flow assays have been successfully used for decades. Nevertheless,  there is a general consensus that the properties of materials used in classic lateral flow assays are difficult to control. However, modern microfluidic devices, besides providing an excellent alternative, can also complement lateral flow assays  with respect to pre-treatment of the clinical sample and transport of small volumes of liquids through micro-channels.

In some of our studies we applied microfluidics as an efficient way to provide reagents and control fluid transport in multistep LF assays. Robust, low-complexity and low-cost systems were designed that integrated the UCP-CF assay format. These devices are well suited for applications in resource poor. Various forms of robust, low-tech microfluidic devices were developed for use outside of well-equipped laboratories. The pressure required for liquid flow can be generated through air pouches that can be actuated either manually, or by a spring-loaded timer, or electronically. This type of disposable self-contained immunoassay cassettes that uses air pouches to propel fluids, requires a sample port and volume metering chamber, storage chambers for reagents, mixing channels, as well as a lateral flow strip for analysis. The developed cassettes perform the assay unattended in consecutive flow format,  but may include complex sample processing as well. The results of the assay are determined by scanning the strip using a handheld battery operated reader. A more complex, high-tech portable microfluidic system was also explored for possibilities of advanced sample preparation, including RNA isolation and nucleic acid amplification. The designed, modular, microfluidic chips allow for simultaneous detection of antibody and nucleic acid following the UCP-SF and UCP-CF format. The explored devices have a generic design such that only the type of LF strip varies with the disease to be diagnosed or (in case of RNA or DNA detection) besides the LF strip also the nucleic acid primers.

Personalized medicine – towards home monitoring of chronic diseases

The use of POC assays as developed by us, is not restricted to the diagnosis of infectious disease. Monitoring of expensive immunodrugs in an individual (personalized medicine) has significant value as well. Example is the monitoring of TNF-α inhibiting therapeutic monoclonal antibodies (anti-TNFs) as infliximab (Remicade®) and adalimumab (Humira®), used for the treatment of autoimmune diseases such as inflammatory bowel disease (IBD) and rheumatoid arthritis (RA). For patients with moderate-to-severe Crohn’s disease (CD), infliximab (IFX) treatment is effective for patients that do not respond to conventional therapy. Treatment with TNF-α inhibiting drugs is very expensive and varies from 15-25 k$/per year/per patient.  However, a significant proportion of initially responding patients loose response due to antibody formation to infliximab and, as consequence, enter a state of inadequate therapeutic drug levels. Currently monitoring of anti-TNFs is rarely applied since only a few laboratories have the tools to accurately perform the required ELISA. Patients are therefore treated according to predetermined protocols. The possibility to measure infliximab levels immediately before drug infusion with a straightforward rapid assay would allow immediate adjustment of the drug level. I preliminary studies we have shown that assays such as the recently developed and successfully evaluated UCP-CF assay for quantitation of IFX serum trough levels this type of testing/monitoring is a reality. We are currently performing research to test the use of finger prick blood and implement the test in the outpatient clinic for the treatment of patients with Crohn’s disease (collaboration with the Department of Gastroenterology, LUMC).


Literature

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  3. Corstjens, P., Zuiderwijk M., Brink A., Li S., Feindt H., Niedbala R. S. and Tanke H.,  "Use of up-converting phosphor reporters in lateral-flow assays to detect specific nucleic acid sequences: A rapid, sensitive DNA test to identify human papillomavirus type 16 infection," Clinical Chemistry 47(10), 1885-1893 (2001).
  4. Zuiderwijk, M., Tanke H. J., Sam N. R. and Corstjens P. L.,  "An amplification-free hybridization-based DNA assay to detect Streptococcus pneumoniae utilizing the up-converting phosphor technology," Clin. Biochem. 36(5), 401-403 (2003).
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  22. Ongagna-Yhombi, S. Y., Corstjens P., Geva E., Abrams W. R., Barber C. A., Malamud D. and Mharakurwa S.,  "Improved assay to detect Plasmodium falciparum using an uninterrupted, semi-nested PCR and quantitative lateral flow analysis," Malar. J. 12 74 (2013). 
  23. van Dam, G. J., de Dood C. J., Lewis M., Deelder A. M., van Lieshout, L., Tanke H. J., van Rooyen L. H. and Corstjens P. L.,  "A robust dry reagent lateral flow assay for diagnosis of active schistosomiasis by detection of Schistosoma circulating anodic antigen," Exp. Parasitol. 135(2), 274-282 (2013).
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  25. Bobosha, K., Elisa M. Tjon Kon Fat, Susan J.F. van de Eeden, Yonas Bekele, Jolien J. van der Ploeg-van Schip, Claudia J. de Dood, Karin Dijkman, Kees L.M.C. Franken, Louis Wilson, Abraham Aseffa, John S. Spencer, Tom H.M. Ottenhoff, Paul L.A.M. Corstjens & Annemieke Geluk. “Field- evaluation of a new lateral flow assay for detection of cellular and humoral immunity against Mycobacterium leprae”, PLoS NTD 8:e2845 (2014).
  26. Corstjens, P.L.A.M., Claudia J. de Dood, Dieuwke Cornelis, Elisa M. Tjon Kon Fat, R. Alan Wilson, Thomas M. Kariuki, Ruth K. Nyakundi, Phillip T. LoVerde, William R. Abrams, Hans J. Tanke, L. van Lieshout, Andre M. Deelder & Govert J. van Dam. Tools for diagnosis, monitoring and screening of Schistosoma infections utilizing lateral-flow based assays and upconverting phosphor labels. Parasitology (2014).
  27. Corstjens, P.L.A.M., Claudia de Dood, Jeff W. Priest, Hans J. Tanke & Sukwan Handali. “A lateral flow test for neurocysticercosis using novel up-converting nanomaterials and a lightweight strip analyzer”, PLoS NTD 8:e2944 (2014).
  28. Wilson, S., Frances M. Jones, Govert J. van Dam, Paul L.A.M. Corstjens, Gilles Riveau, Colin M. Fitzsimmons, Moussa Sacko, Birgitte J. Vennervald & David W. Dunne. “Human Schistosoma haematobium anti-fecundity immunity is dependent on transmission intensity and is associated with IgG1 to worm-derived antigens”, J. Infect. Dis. (2014).
  29. van Dam, G.J., Jing Xy, Robert Bergquist, Claudia J. de Dood, Jürg Utzinger, Zhi-Qiang Qin, Wei Guan, Ting Feng, Xin-Ling Yu, Jie Zhou, Ma Zheng Xiao-Nong Zhou & Paul L.A.M. Corstjens. “An ultra-sensitive assay targeting the ciruculating antodic antigen for the diagnosis of Schistosoma japonicum in a low-endemic area, People’s Republic of China”, Acta Tropica September 6th (2014).
  30. van Dam, G.J., Peter Odermatt, Luz P. Acosta, Robert Bergquist, Claudia J. de Dood, Dieuwke Kornelis, Jürg Utzinger & Paul L.A.M. Corstjens. “Evaluation of banked urine samples for the detection of circulating anodic and cathodic antigens in Schistosoma mekongi and S. japonicum”, Acata Tropica (2014).