Bright-field and fluorescence chip-scale microscopy for biological imaging

Optical microscopy is an essential tool in biological science and one of the gold standards for medical examinations. Miniaturization of microscopes can be a crucial stepping stone towards realizing compact, cost-effective and portable platforms for biomedical research and healthcare. This thesis reports on implementations of bright-field and fluorescence chip-scale microscopes for a variety of biological imaging applications. The term “chip-scale microscopy” refers to lensless imaging techniques realized in the form of mass-producible semiconductor devices, which transforms the fundamental design of optical microscopes.

Our strategy for chip-scale microscopy involves utilization of low-cost Complementary metal Oxide Semiconductor (CMOS) image sensors, computational image processing and micro-fabricated structural components. First, the sub-pixel resolving optofluidic microscope (SROFM), will be presented, which combines microfluidics and pixel super-resolution image reconstruction to perform high-throughput imaging of fluidic samples, such as blood cells. We discuss design parameters and construction of the device, as well as the resulting images and the resolution of the device, which was 0.66 µm at the highest acuity. The potential applications of SROFM for clinical diagnosis of malaria in the resource-limited settings is discussed.

Next, the implementations of ePetri, a self-imaging Petri dish platform with microscopy resolution, are presented. Here, we simply place the sample of interest on the surface of the image sensor and capture the direct shadow images under the illumination. By taking advantage of the inherent motion of the microorganisms, we achieve high resolution (~1 µm) imaging and long term culture of motile microorganisms over ultra large field-of-view (5.7 mm × 4.4 mm) in a specialized ePetri platform. We apply the pixel super-resolution reconstruction to a set of low-resolution shadow images of the microorganisms as they move across the sensing area of an image sensor chip and render an improved resolution image. We perform longitudinal study of Euglena gracilis cultured in an ePetri platform and image based analysis on the motion and morphology of the cells. The ePetri device for imaging non-motile cells are also demonstrated, by using the sweeping illumination of a light emitting diode (LED) matrix for pixel super-resolution reconstruction of sub-pixel shifted shadow images. Using this prototype device, we demonstrate the detection of waterborne parasites for the effective diagnosis of enteric parasite infection in resource-limited settings.

Then, we demonstrate the adaptation of a smartphone’s camera to function as a compact lensless microscope, which uses ambient illumination as its light source and does not require the incorporation of a dedicated light source. The method is also based on the image reconstruction with sweeping illumination technique, where the sequence of images are captured while the user is manually tilting the device around any ambient light source, such as the sun or a lamp. Image acquisition and reconstruction is performed on the device using a custom-built android application, constructing a stand-alone imaging device for field applications. We discuss the construction of the device using a commercial smartphone and demonstrate the imaging capabilities of our system.

Finally, we report on the implementation of fluorescence chip-scale microscope, based on a silo-filter structure fabricated on the pixel array of a CMOS image sensor. The extruded pixel design with metal walls between neighboring pixels successfully guides fluorescence emission through the thick absorptive filter to the photodiode layer of a pixel. Our silo-filter CMOS image sensor prototype achieves 13-µm resolution for fluorescence imaging over a wide field-of-view (4.8 mm × 4.4 mm). Here, we demonstrate bright-field and fluorescence longitudinal imaging of living cells in a compact, low-cost configuration.

Study of the effect of alcohol on single human red blood cells using near-infrared laser tweezers Raman spectroscopy

The effect of alcohol solution on single human red blood Cells (RBCs) was investigated using near-infrared laser tweezers Raman spectroscopy (LTRS). In our system, a low-power diode laser at 785 nm was applied for the trapping of a living cell and the excitation of its Raman spectrum. Such a design could simultaneously reduce the photo-damage to the cell and suppress the interference from the fluorescence on the Raman signal. The denaturation process of single RBCs in 20% alcohol solution was investigated by detecting the time evolution of the Raman spectra at the single-cell level. The vitality of RBCs was characterized by the Raman band at 752 cm(-1), which corresponds to the porphyrin breathing mode. We found that the intensity of this band decreased by 34.1% over a period of 25 min after the administration of alcohol. In a further study of the dependence of denaturation on alcohol concentration, we discovered that the decrease in the intensity of the 752 cm(-1) band became more rapid and more prominent as the alcohol concentration increased. The present LTRS technique may have several potential applications in cell biology and medicine, including probing dynamic cellular processes at the single cell level and diagnosing cell disorders in real time. Copyright (c) 2005 John Wiley T Sons, Ltd.

Rhythms of division of some algae in situ and in laboratory culture. [Translation from: Studii Cerc.Biol.(Bot.) 21, 61-65, 1969. ]

The rhythm of division of 9 species belonging to different groups of algae were analysed in situ and in the laboratory. The research which developed in different environmental conditions attempted to establish the capacity for multiplication and assimilation of chlorophyll on the part of the algae under study with a view to placing them in a culture. The results obtained showed that the green multicellular algae (eg. Ulothrix) and the blue algae (eg. Lyngbya, Oscillatoria) are able to produce an appreciable quantity of dry matter, just as the unicellular algae. At the same time it arises that amongst the numerous factors of the environment, temperature plays one of the most important roles in the process of multiplication.

The changes in various iron compounds in culture media for algae. [Translation from: Archiv fur Hydrobiologie Supplemente Band 38(1/2) 151-169, 1970.]

The purpose of this work is a contribution to the quantitative record of the use of iron by planktonic algae. Preliminary experiments with Chlorella to determine the rate of iron intake in the presence of inorganic sources of iron did not produce the desired result. The crucial point of this work is the investigation of the influence of various external factors on the stability of FeEDTA (FeEDTA = Ferric(III)-compound of ethylene-diamine tetra-acetic acid), since this compound appears to be particularly well-suited as a source of iron for planktonic algae (e.g. TAMIYA et al. 1953). Cultures of Chlorella fusca in a light thermostat were used in experimental research. Methods and results are discussed.

Special carbon requirement of Spirulina platensis culture. [Translation from: Materialy VII Vsesoyuznogo Rabochego Soveshchanii po Voprosu Krugovorota Veshchestv v Zamknutoi Sisteme na Osnove Zhiznedeyatel'nosti Nizshikh Organizmov p55-57. Kiev, Naukova Dumka, 1972.]

By the industrial cultivation of blue-green algae, there very much appears the important question about their carbon nutrition. Spirulina grows within the range of pH value of medium of 8.5 - 11.0. In this range of pH value in the culture medium CO2 is present in the form of bicarbonate and carbonate, which serves as principal source of carbon for the present type of algae. There is little information yet about the influence of the pH of the medium, and the form of carbon components of the medium, on the rate-increase of Spirulina. Investigations were conducted into the influence of some pH values of medium on the rate-increase of the alga Spirulina platensis.

Experimental culture of algal balls (Cladophora sauteri). Retention of original shape [Translation from: Bull.Jap.Soc.Phycol. Vol. 10, (1), p. 23-27, 1962]

It was on July 1960 when 10 algal balls were acquired for exhibition at Suma Aquarium, Kobe. Permission to remove the specimens from the Lake Akan Reserve was given by the National Nature Reserve Committee. Algal balls, as a rule, lose their natural beauty when they are kept in an ordinary tank for a certain length of time. In an effort to retain the natural beauty it was decided to exhibit them in culture. This paper summarises the findings of this experiments with Cladophora sauteri. The author concludes that serious consideration has to be given as to the intensity of light, the sunlight, the water temperature and the nutrition for algal balls in culture in order to retain the natural beauty and shape.

The culture of algae and its applications

An article explaining how the methods and results from the time spent by the author culturing algae can be applied to other algal investigations. The work by the author found that physiological requirements differ widely among algae belonging to different systematic groups. Details are given of the results of a series of experiments which were undertaken in solutions with similar proporties to some natural waters in the Lake District. Reference is made to a paper under preparation at that time containing data on phytoplankton studied in the field within the Lake District during 1937. Reference is also made to Loch Leven and the affects of bluegreen alga on the number of trout caught weekly during 1937.

Delivery of Targeted Nanoparticles Across the Blood-Brain Barrier Using a Detachable Targeting Ligand

Chronic diseases of the central nervous system are poorly treated due to the inability of most therapeutics to cross the blood-brain barrier. The blood-brain barrier is an anatomical and physiological barrier that severely restricts solute influx, including most drugs, from the blood to the brain. One promising method to overcome this obstacle is to use endogenous solute influx systems at the blood-brain barrier to transport drugs. Therapeutics designed to enter the brain through transcytosis by binding the transferrin receptor, however, are restricted within endothelial cells. The focus of this work was to develop a method to increase uptake of transferrin-containing nanoparticles into the brain by overcoming these restrictive processes.

To accomplish this goal, nanoparticles were prepared with surface transferrin molecules bound through various liable chemical bonds. These nanoparticles were designed to shed the targeting molecule during transcytosis to allow increased accumulation of nanoparticles within the brain.

Transferrin was added to the surface of nanoparticles through either redox or pH sensitive chemistry. First, nanoparticles with transferrin bound through disulfide bonds were prepared. These nanoparticles showed decreased avidity for the transferrin receptor after exposure to reducing agents and increased ability to enter the brain in vivo compared to those lacking the disulfide link.

Next, transferrin was attached through a chemical bond that cleaves at mildly acidic pH. Nanoparticles containing a cleavable link between transferrin and gold nanoparticle cores were found to both cross an in vitro model of the blood-brain barrier and accumulate within the brain in significantly higher numbers than similar nanoparticles lacking the cleavable bond. Also, this increased accumulation was not seen when using this same strategy with an antibody to transferrin receptor, indicating that behavior of nanoparticles at the blood-brain barrier varies depending on what type of targeting ligand is used.

Finally, polymeric nanoparticles loaded with dopamine and utilizing a superior acid-cleavable targeting chemistry were investigated as a potential treatment for Parkinson’s disease. These nanoparticles were capable of increasing dopamine quantities in the brains of healthy mice, highlighting the therapeutic potential of this design. Overall, this work describes a novel method to increase targeted nanoparticle accumulation in the brain.

Fast and Efficient Neural Conversion of Human Hematopoietic Cells

Neurons obtained directly from human somatic cells hold great promise for disease modeling and drug screening. Available protocols rely on overexpression of transcription factors using integrative vectors and are often slow, complex, and inefficient. We report a fast and efficient approach for generating induced neural cells (iNCs) directly from human hematopoietic cells using Sendai virus. Upon SOX2 and c-MYC expression, CD133-positive cord blood cells rapidly adopt a neuroepithelial morphology and exhibit high expansion capacity. Under defined neurogenic culture conditions, they express mature neuronal markers and fire spontaneous action potentials that can be modulated with neurotransmitters. SOX2 and c-MYC are also sufficient to convert peripheral blood mononuclear cells into iNCs. However, the conversion process is less efficient and resulting iNCs have limited expansion capacity and electrophysiological activity upon differentiation. Our study demonstrates rapid and efficient generation of iNCs from hematopoietic cells while underscoring the impact of target cells on conversion efficiency.

Análise de uma heme oxigenase funcional em Trypanosoma cruzi

O Trypanosoma cruzi é o agente etiológico da doença de Chagas, transmitida através de insetos vetores triatomíneos durante a alimentação no hospedeiro vertebrado. Os triatomíneos ingerem numa única alimentação cerca de 10 mM de heme ligado à hemoglobina. O heme é uma importante molécula no metabolismo dos organismos. Um mecanismo intracelular importante no controle de sua homeostase é a degradação enzimática pela Heme Oxigenase (HO) formando biliverdina (Bv), monóxido de carbono e ferro. Como esta enzima não está presente no genoma de T. cruzi, esse trabalho tem por objetivo identificar uma atividade funcional de HO neste parasito, uma vez que dados do nosso laboratório mostram a presença de biliverdina nas incubações dessas células com heme. No presente trabalho testamos o efeito do SnPPIX (inibidor da HO-1), CoPPIX (indutor da HO-1) e Bv sobre a proliferação da forma epimastigota do parasito. A adição de SnPPIX diminuiu a proliferação do parasito na tanto na ausência quanto na presença de heme. Quando a Bv foi adicionada à cultura esse efeito foi revertido; a Bv aumenta a proliferação celular na presença de heme. Por outro lado, a adição de CoPPIX não interferiu na proliferação. Posteriormente, mostramos através da técnica de immunoblotting, utilizando anticorpo monoclonal contra a HO-1, um aumento da expressão de uma proteína em resposta ao heme. Diferentemente das HO-1 já descritas que possuem massa molecular de 32 kDa, a única banda reconhecida pelo anticorpo apresenta 45 kDa. Analisamos também a expressão da HO-1 na presença de CoPPIX, SnPPIX e biliverdina, e somente o CoPPIX foi capaz de modular os níveis de expressão da HO-1. A análise estrutural através da técnica de imunocitoquímica mostrou uma maior expressão da enzima na presença de heme, e que a HO-1 de T. cruzi pode ter mais de uma localização, apresentando marcação citoplasmática e glicossomal. A fim de investigar a sequência da HO-1 de T. cruzi, o DNA genômico foi extraído para amplificação por PCR do gene da HO-1 utilizando oligonucleotídeos desenhados no genoma de T. cruzi. Os dois pares de oligonucleotídeos utilizados nao foram capazes de amplificar uma sequência equivalente a uma HO. Em seguida, utilizamos a técnica de imunoprecipitação, seguida de immunoblotting, com anticorpo anti-HO-1, com objetivo de concentrar a proteína alvo, e observamos um aumento significativo do imunocomplexo nas células tratadas com heme 300 mM, cerca de 2 vezes em relação ao controle. Dando seguimento à tentativa de identificação da HO-1 de T. cruzi, utilizamos a técnica de espectrometria de massa a partir de eletroforese unidimensional, que mostrou uma grande alteração do perfil protéico na presença de heme, mas futuros experimentos são necessários, como eletroforese 2D, para a identificação da proteína alvo

A behavior model for blood donors and marketing strategies to retain and attract them

Objective: analyze and propose a theoretical model that describes blood donor decisions to help staff working in blood banks (nurses and others) in their efforts to capture and retain donors. Methods: analysis of several studies on the motivations to give blood in Spain over the last six years, as well as past literature on the topic, the authors' experiences in the last 25 years in over 15 Non Governmental Organizations with different levels of responsibilities, their experiences as blood donors and the informal interviews developed during those 25 years. Results: a model is proposed with different internal and external factors that influence blood donation, as well as the different stages of the decision-making process. Conclusion: the knowledge of the donation process permits the development of marketing strategies that help to increase donors and donations.

Farming experiments and transfer of technology of bivalve culture along the southwest coast of India

The Central Marine Fisheries Research Institute (CMFRI) in India developed bivalve farming technologies in the 1970s, but these were not widely adopted at the time. In 1993, CMFRI undertook an action research program to encourage farming of edible oysters (Crassostrea madrasensis), mussels (Perna viridis and Perna indica), clams (Paphia malabarica) and pearls (Pinctada fucata) along the southwest coast of India. Successful demonstration of the viability of bivalve farming led to the initiation of commercial farming of mussels and generated interest among farmers and entrepreneurs in developing production of pearls and farming of edible oysters. Given the high potential for the mollusc aquaculture, both for the local and export market, issues such as demarcation and issuance of lease right on aquaculture zones in public waterbodies by the government, organization of marketing systems and provisions for technical and financial support to farmers need to be addressed.

Culture of tropical sea cucumbers for stock restoration and enhancement

Severe overfishing of sea cucumbers has occurred in most countries of the tropical Indo-Pacific. The release of juveniles is being examined at the ICLARM Coastal Aquaculture Centre in the Solomon islands as a means of restoring and enhancing tropical sea cucumber stocks. Sandfish (Holothuria scabra) are the tropical species that show the best potential for stock enhancement. Sandfish are of high value, widely distributed and relatively easy to culture in simple systemss at a low cost. This paper summarizes information about the culture of H. scabra and compares it to that of the temperate species Stichopus japonicus.

Estudo exploratório das iniciativas acerca da segurança do paciente em hospitais do Rio de Janeiro

Trata-se de um estudo descritivo e exploratório, que se apoiou na estatística descritiva para abordagem dos resultados produzidos. Tem como objeto as iniciativas para segurança do paciente, implementadas pelos gerentes de risco em hospitais do município do Rio de Janeiro. O estudo teve como objetivo: analisar as iniciativas implementadas pelos gerentes de risco para garantir a segurança do paciente, considerando as iniciativas nacionais e mundiais existentes. Foi desenvolvido em cinco hospitais do Rio de Janeiro, com quatorze gerentes de risco. A técnica utilizada foi a aplicação de um questionário semiestruturado, composto por questões fechadas e abertas sobre as iniciativas para segurança do paciente. Foi verificado que todos realizam atividades voltadas para educação continuada. As menos desenvolvidas são ações de tecno, hemo e farmacovigilância (29%). A maioria informou que se orienta pela Agência Nacional de Vigilância Sanitária, assim como implementa quatro programas para segurança do paciente: a identificação dos pacientes (100%), seguida da assistência limpa é uma assistência mais segura (86%), controle de infecção da corrente sanguínea associada ao cateter (64%) e cirurgia segura, salva vidas (64%). A maior parte dos gerentes de risco desconhece os cinco protocolos operacionais padronizados da Joint Comission on Acreditation of Healthcarecare Organizations e o conteúdo da campanha dos 5 milhões de vidas do Institute for Healthcare Improvement. Os eventos adversos cujo monitoramento é prioritário para os gerentes de risco, são queda do leito (43%) e infecções (36%). A maior parte deles (57%) informa utilizar a análise de causa raiz e análise do modo e efeito da falha como ferramentas de monitoramento de eventos adversos. Conclui-se que grande parte das iniciativas para segurança do paciente são implementadas pelos gerentes de risco, o que vai ao encontro do que é sugerido atualmente, no entanto as iniciativas mais citadas são as iniciativas já divulgadas pelas instituições de referência para segurança do paciente, e que exigem poucos investimentos para serem implementadas, logo é essencial mais ações de capacitação dos gerentes de risco e de desenvolvimento de uma cultura de segurança no ambiente hospitalar.