лебедева

GE Healthcare

Life Sciences

“Together, we make the world healthier through imaginative science"

Технология мониторинга экспрессии клеточных геномов

Евгения

Чакина

ООО

«Аламед»Официальный

дистрибьюторGE Healthcare Life

Sciences

Лебедева Л., IV Всероссийская школа по клинической иммунологии, 2013г.

Развитие

исследований: от

гена

до

функции

Genomics

Секвенирование

генов,

экспрессионный

анализ,

сайт‐специфический

мутагенез

и

др.

Proteomics

Идентификация

и

характеризация

белков,

анализ

их

модификаций

и

взаимодействий

и

др.

Ettan

DIGE

DNA Microarray

8.48E10 0

1 35 2 3 3

C - 12C - 2C - 4C - 5C - 6C - 7

C - 3C - 8D - 3B - 2B - 3D - 2C - 9D - 1B - 4B - 6B - 5D - 7

B - 7B - 1B - 8D - 9B - 9B - 12B - 11D - 6D - 11C - 10C - 11

D - 10B - 10D - 8D - 12D - 5D - 4

Cellomics

Исследование

клеточных

функций, локализации

молекул

и органелл, клеточного

фенотипа

и

др.

YFP-H2B in Arabidopsis thaliana

protoplasts


Выступающий

Заметки для презентации

After completion of the sequencing of the the human genome we are now entering the postgenomic age. The main focus in genomic research is switching from sequencing to using the genome sequences in order to understand how genomes are functioning. Although we tend to take our current technologies for granted, sometimes it’s helpful to take a step back and appreciate that over the past decade or so we’ve experienced an enormous revolution in the way we do science. No longer are we confined to looking at genes one at a time in isolation, instead a wave of new advances in sequencing and microarray technologies have enabled us to investigate nucleic acids on a genome-wide scale. The same is true with proteins—we now have robust technologies to examine entire proteomes en masse. The latest advance is to extend this concept to the cell level, where we can examine the function and behavior of genes and proteins in a more physiologically relevant context. The leading technology that has made this possible is called “high content analysis”.

Структурная

и

функциональная протеомика

Задачи

исследователя:•

Определение

структуры

белка

•


функций

белка

/ антитела•

Регуляция

•

Межмолекулярные

взаимодействия/активность•

Биофармакология

Этапы:•

Пробоподготовка

‐

Очистка

белка

•

Разделение

с

высоким

разрешением

/ Анализ

cтруктуры•

Анализ

функции


Структурная

протеомика 2 разных

подхода

LC‐MS: смесь

белков

→ определение

пептидов

в

смеси

•

Разделение

предварительно

очищенного

образца методом

ВЭЖХ

или

УльтраВЭЖХ

•


структуры

методом

MS

2D‐MS:

один

белок

→ определение

структуры

белка

•

Фракционирование

образца

методом

2D‐фореза, поиск

интересующих

белков

(DIGE), извлечение

единичных

пятен

–

молекул

белков•


структуры

методом

MS


Функциональная

протеомикаФундаментальные

исследования

взаимодействий:

•

Изучение

кинетики

и

аффинности


молекул•

Задачи:

изучение

каскадов

реакций, функций белков,

формирования

белковых

комплексов

Фишинг:

смесь

белков

→

«вылавливание

единичной

молекулы»

→

определение

структуры

•

Получение

фракций

на

FPLC•

Скрининг

фракций

на

чипе

–


взаимодействующей

молекулы•


структуры

методом

MS

Фармацевтические


и

биотехнология:•

Скрининг

кандидатов

в

фармпрепараты

и

антител

для

диагностикумов•

Персонализированная

медицина

–


эффективности

и

метаболизма

лекарственных

средств


Двумерный

электрофорез

2D


–

незаменимый

классический

метод

в протеомных

исследованиях, «входной

билет

в

протеомику»

Технология

метода

основана

на

последовательном разделении

белковых

образцов

в

двух

направлениях: по

изоэлектрической

точке

и

массам

молекул.

Модификация

метода

2D‐электрофореза

–

технология

2D‐DIGE

(анализ

дифференциально

окрашенных

гелей)‐

существенно

сокращает

временные

и

финансовые

затраты

за

счет

мультиплексирования

‐ повышает точность, достоверность

и

воспроизводимость

результатов

за счет

унифицированных

условий

эксперимента

и

использования

внутреннего

стандарта.


2D‐электрофорез: этапы.

MS


Двумерный


–

результат?Лебедева Л., IV Всероссийская школа по клинической иммунологии, 2013г.

2‐D DIGE для

экспрессионной протеомики

Традиционный

2D электрофорезТрудозатратный, большие

вариации

внутри

эксперимента

(одна

краска

для

пост‐окрашивания,

требуются

повторные

эксперименты

для

разделения биологических

и

технических

вариаций)

Технология

DIGE Обеспечивает

большую

точность, воспроизводимость

и

существенно

сокращает

трудозатраты

и

время

эксперимента:

•

Мультиплексирование

–

несколько

образцов

анализируются

в

одном геле

•

«Безобразно

но

однообразно» – ошибка

оператора

будет

одинакова для

всех

образцов

в

геле

•

Внутренний

стандарт

–

присутствует

во

всех

гелях

в

одном

эксперименте


Difference gel electrophoresis (DIGE)Лебедева Л., IV Всероссийская школа по клинической иммунологии, 2013г.

Cy3 Mouse

proteins

control

Cy2 Mouse liver proteins

Internal standard

Overlay Mouse liver proteins

Cy5 Mouse liver proteins

treated


8 образцов: 8 гелей

x 3 повтора

= 24 геля

2D‐DIGE (3‐цветный)


2‐D и

2‐D DIGE


2D электрофорез

(1‐цветный)

8 образцов:4 геля

(без

повторностей) = 4 геля


Функциональная протеомика


Biacore™

‐

биосенсоры

SPR

MicroCal™

‐ калориметр

Анализ

взаимодействия

молекул:

‐ Без метки‐

В

режиме

реального

времени

‐

Кинетика



Анализ


различных молекул:

•

Белки

•

Нуклеиновые

кислоты

•

Липиды

и

мембран‐ассоциированные

молекулы

•

Углеводы

•

Низкомолекулярные

соединения

•

Целые

клетки

•

Вирусы/бактерии




This slide illustrates that Biacore assays can be used for a wide range of molecules, e.g. proteins, nucleic acids, lipids & membrane associated molecules, carbohydrates, LMW (Low Molecular Weight) compounds (≤ 100 Da), whole cells, viruses and bacteria.

Biacore. SPR. Используются

в

ведущих

академических

и

биотехнологических

лабораториях

с

1990

г.

Система детекции

SPR

‐

BiacoreTM

Сенсорный чип

Программное обеспечение

Микрофлюид/ проточные

ячейки




Biacore is a fully integrated system which combines Surface plasmon resonance (SPR) detection technology, which monitors interactions on the chip surface in real time without labels. Sensor chip surfaces – the interaction and detection occur on the chip surface that forms one wall of the flow cell. One interactant is immobilized on the chip surface and the other is injected in solution. Microfluidics – Biacore has a continuous flow system where sample is injected at a constant flow rate. User-friendly software for control and evaluation of results.

Сенсограмма

SPR

Базовая

линия

буфер образец

ассоциация

буфер

диссоциация

Точка

отсчета

Ответсвязывания

Форма

кривой

показываеткинетику


•

Специфичность

•

Аффинность

•

Кинетика

•

Концентрация

•

Термодинамика




Animated build to explain principles of labe-free interaction analysis with Biacore systems The sensorgram provides real-time information about the entire interaction, with binding responses measured in resonance units (RU). Binding responses at specific times during the interaction can also be selected as report points. A schematic figure shows an example of a sensorgram i.e. protein interaction. You start with a biomolecule captured onto your sensorchip surface. Buffer flows over the surfacce to establish a baseline. Adding your sample a change a mass occurs as it binds to the capture molecule detected by the optical unit. Sample addition is stopped and changed to a buffer flow again, you then see molecule dissociation in real time. Specificity analysis Is my molecule of interest specific for its target? Flexibility in Biacore™ assay design allows assessment of cross-reactivity and specificity Examples: Does my antibody recognize multiple derivatives? Which members of the protein family are detected? Screen for inhibitor specificity by using several ligands Determine the specificity of a drug for its target receptor Concentration analysis How much analyte is there in my sample? Examples: Serum concentration of IgG Quality control of production batches Levels of streptomycin in bovine milk Affinity analysis How strong is the binding between analyte and ligand? Usually used when the association and dissociation rates are too fast to determine accurately Examples Affinity of a monoclonal antibody for its antigenic epitope Affinity of the T cell receptor for MHC class II Affinity of a drug for its target receptor Kinetic analysis How fast do the ligand and analyte bind (associate) and how fast does the complex fall apart (dissociate)? Kinetic data reveal more information than affinity Interactions with the same affinity may have different kinetic characteristics Thermodynamics Temperature dependence of kinetics and affinity Reactions are generally faster at higher temperatures Affinity may go either way as the temperature changes Thermodynamics in Biacore™ analyzes the temperature dependence Driving force for the reaction/interaction Type of interaction involved Structure-function relationships

В шприце ‐ лиганд (связываемое

соединение)

В

экспериментальной

ячейке

‐

целевой

белок

В

ячейке

сравнения

‐

буфер

Измеряется

теплота


Обработанныеданные

Данныеэксперимента

MicroCal™

‐

принцип

Ячейкасравнения

Экспериментальнаяячейка

Шприц

-14

-12

-10

-8

-6

-4

-2

0

kcal

/mol

e of

inje

ctan

t

0 1 2Molar Ratio

СтехиометрияАффинность

Механизм


Около

20 лет

на

рынке

–

более

10 000 научных

публикаций

•

Онкология

•

Нейробиология

•

Иммунология

•

Инфекционные

заболевания

•

Протеомика

•

Трансдукция

сигнала

•

Разработка

вакцин, фармпрепаратов

и

диагностикумов

•

Выбор

и

характеристика

связывающих

реагентов

•

Исследования

лекарств

и

многое

другое….




High Content Analysis (HCA) это…

‐

Анализ

«высокого

содержания»

‐

Получение

большого

количества разноплановых

данных

по

одному

образцу

‐

Многопараметрический

анализ

данных




High content analysis, or HCA, is the ability to extract and make sense of multi-parameter data from high-throughput, sub-cellular imaging. Taking more than one piece of meaningful data from assays in a cellular environment. Workflow: RAPIDLY imaging cells multiwell plates or slides, EXTRACTING data from the images using sophisticated analysis software, ANALYZING that data to better understand the function and behavior of target genes and proteins.

Эволюция

клеточного

анализа

Деструктивный

Электрофорез

Фиксированный


сигнала

Динамический

Процессы

в

клетке

Интегрированный

Взаимодействия

Инф

орма‐

Тивн

оcть


Зачем

нужен

HCA?

Исследования:

•

Изучение

функции,

локализации

и

кинетики

в

живой клетке:

транслокации, активация

рецепторов, фазы

клеточного

цикла•

Тестирование

воздействия

внешних

условий

(токсинов, лекарственных

средств, ингибиторов, активаторов

и

пр.) на

живую

клетку

•

Слежение

за

несколькими

морфологическими параметрами

• Возможность

мечения

нескольких

мишеней

• Сложные

многопараметрические

эксперименты•Мультиплексность•Широкомасштабные

скрининговые


• Быстрота

получения

данных• Огромное

количество

данных

по

одному

образцу

• Сохранение

изображений

и

повторные

анализы


Cytometry

Western blotting

Универсальность и возможности

Электро

форез

Цитофлуориметр

Микроскопия




Many customers find that they are able to use a HCA system to achieve comparable results to what they could achieve with more traditional technologies. At the same time, they get the added benefit of information-rich images, so that they can ask even more complex questions. A HCA system does not by any means replace these more traditional technologies, but rather it complements existing technologies by providing more information in a functional context. Results gained from any of these techniques can be further explored in a functional way using HCA. Conversely, in many cases it is helpful to follow up results obtained with a HCA system using techniques like western blotting and cytometry. Manual Microscopy – HCA brings microscopy based studies, increased productivity with less hands on time with it’s automated image capture and non subjective image analysis software. Manual scoring using a microscope is very time consuming and our validated, user friendly, preconfigured software takes the labour out of writing an analysis routine for 90% of cellular events. (For very complex analysis, Developer is a specialised and highly powerful tool using the same workflow) Flow Cytometry – provides statistical analysis of cell populations using fluorescence intensity. HCA compliments this technique by providing intensity, morphology, localisation and movement data on populations of interest down to the single cell level in a NON DESTRUCTIVE way. Western Blotting – Although Western Blotting and HCA are not directly comparable, most Western Blotters are likely to be interested in the cellular function of the protein they are studying. Traditionally researchers often use a combination of blotting and manual microscopy once a protein has been isolated and profiled. However, if they are studying expression, localisation or modification of proteins in cella then doing the work ‘in situ’ in side the cell is a far more efficient approach – see the Bristol University example later on.

Основные

компоненты

HCA

РеактивыФлуоресцентные

метки

ОборудованиеИнструмент

для

визуализации

фиксированных и живых клетокДополнительные

модули

(инкубатор, система

дозирования) для

прижизненного

наблюдения

Программное

обеспечениеДля

получения,

обработки

и

хранения

HCA данных

Биологический

объектКультуры

клеток

ТканиОрганизмы




In recent years, technological advances in these components have come together to make HCA possible. These are the main components to consider if you want to adopt HCA. The quality of each of the elements will have significant impact on the quality and utility of the output. Talk about reagents e.g GFP, Cy Dyes, Cell factory (stable, transient and non-engineered)

IN Cell Analyzer 2200, 6000Лебедева Л., IV Всероссийская школа по клинической иммунологии, 2013г.

Высоко‐производительная

камера

Стандартная

CCD-камера1.4 Mp CoolSNAP

ES21392 x 1040 пикс.6.45мкм

кв. пикс.137 клеток

при

10X

увеличении

Широкоформатная

камера4.2 Mp CoolSNAP

K4;2048 x2048 пикс.7.40мкм

кв. пикс.635 клеток

при

10X

увеличении

Больше

клеток, больше

данных, быстрее!!!


Новейшая

технология

восстановленияизображения

(Деконволюция)




The IN Cell Analyzer 2000 imaging platform offers advanced image restoration options to ensure you get the most quantitatively accurate images possible from the system. All lens systems (from microscopes to the Hubble telescope! Chose this method over ‘Nipkow disc tupe’ confocality because of ability to use all light available with this method) introduce some degree of blurring during image generation. In the case of fluorescence microscopes, it is possible to accurately model the blurring function associated with the objective lens, and apply this function to recover the original object information. Unlike confocal approaches, which discard light emanating from above and below the focal plane, image restoration actually works to recover the original information, making maximum use of signal from dim samples. Image restoration is based on a realistic model of the lens system, and therefore provides more accurate results than image enhancement techniques like ‘nearest neighbour de-blurring’ and ‘sharpening’ provided by commercial image editing packages. The image restoration options available with IN Cell Analyzer 2000 are capable of removing noise, increasing contrast, and improving resolution in both the lateral (X-Y) and axial (Z) dimensions. Deconvolution options in both 2D and 3D acquisition modes greatly increase image accuracy, while Z-axis projection provides a virtually extended depth of field to increase lateral resolution without missing features that fall above or below the focal plane. This is particularly useful when working with high magnification, high numerical aperture lenses, which provide very thin optical sections

http://upload.wikimedia.org/wikipedia/commons/1/12/Improvement_in_Hubble_images_after_SMM1.jpg

3 режима

проходящего

света-микроскопия

светлого

поля-фазово-контрастная

микроскопия-ДИК

(дифференциально-

интерференционныйконтраст)

для

анализа

живых

клеток

2D 2D Deconvolved 2.5D Deconvolved

Использование

опции

3D/деконволюциидля

улучшения

качества

изображения




Whether you need transmitted light images for analysis, or simply want to include them in a publication, the IN Cell Analyzer 2000 imaging platform offers a range of modes to ensure you get the highest quality images possible for your samples. Choose from Brightfield, Differential Interference Contrast (DIC) or Phase Contrast mode. Transmitted light imaging allows label-free monitoring of cell health, movement, morphology and growth, and can be particularly beneficial for extended time-course studies where fluorescent markers could have a toxic effect. For example, the nuclear dye Hoechst interferes which chromosome segregation, and therefore would not be suitable for live cell lineage or cell cycle analysis. The high quality of transmitted light images, combined with advanced image analysis tools for segmentation available in IN Cell Investigator, make label-free cell quantification possible. Transmitted light images can also be analyzed in combination with images acquired in fluorescent channels for added information. Bright field analysis using phase contrast images can be used for cell counting and morphological analysis for cell health and cell confluency We have comparative data obtained for bright analysis versus fluorescent assay analysis ..for example apoptosis detection and neurite outgrowth The bright field imaging and analysis can be performed on fixed or live cells Bright field imaging can be readily multiplexed with fluorescent images

Данные

от

одной

клетки

до

популяцииЛебедева Л., IV Всероссийская школа по клинической иммунологии, 2013г.



Automated linking between IN Cell Investigator Software and Spotfire allows automatic import of data from Investigator analysis into Spotfire for visualisation. Data links between the two applications are maintained allowing dynamic interrogation of your data either at summary or single cell level. Clicking on a data point on a plot, a value in a data table or a cell in an image will highlight all the information relevant to your selection. Here Spotfire is displaying a heat map for nuclear morphology data; each column in the heatmap represents an individual parameter, with each row across the heatmap showing the data from a single cell. Clicking on a row in the heatmap highlights the relevant cell in the image on the right and the data in the data table on the left

Spotfire

–

интерактивное

получение


Движение

клеток

Рост

клеток

Клеточная токсичность

Рост

клеток




Some examples of Spotfire visualisation of complex temporal data sets. Cell tracking plots show output from IN Cell Investigator with cell ID numbers (left) with position of each cell in each time lapse frame indicated and colour coding of cell tracks with time (right). Cell growth plot on left was produced by importing xy coordinates for each cell for each frame and plotting in 3D plot versus time. All done automatically by import from raw data in Developer into Spotfire (cell position in image on x/y axis with position in time shown on Z axis). Time lapse video of Halotag-TMR labelled cells in IN Cell environmental chamber for 36 hours. Plot show increase in cell number over 36 hours in IN Cell Analyzer enviromental chamber. Plot is bottom right is alternative visualisation of same data (in both plots data points are coloured coded to highlight changes in nuclear morphology parameters. Plot in top right is from tox assay live cell time course using PI and Annexin V. On plot x/y coordinates relate to well position of a 384 well plate. Z-axis is time. Animation shows filtering of data in Spotfire to highlight toxic compounds and time at which toxicity occurs.

Области

применения

•

Активация

рецептора

•

Токсикологический

ответ

клетки

и

апоптоз

•


внутриклеточного

сигнала

•

Анализ

дифференцировки

стволовых

клеток

•

Клеточный

цикл

•

Органеллы

и

внутриклеточный

транспорт

•

Функционирование

нейронов

•

Морфологическое

и

физиологическое

состояние

клетки


Данные

пользователей

на IN Cell Analyzer

Human Neuronal Stem Cells from fetal cortex. Cell stained

for DNA (blue), TUJ-1 (neuronal differentiation marker, green) and GFAP (astrocyte

differentiation marker, red).

Courtesy of Kymmy

Lorrain, Brain Cells Inc

NIH-3T3 fibroblasts overexpressing

src

causing localization of actin

toward podosomes. Cells stained for DNA (blue), actin

(green) and phospho-src

(red). Courtesy of Evelyn Griffin, Scripps Florida

Primary Porcine Trabecular

Meshwork cells stained for DNA (blue), actin

(rhodamine

phalloidin, red) and Paxillin

in focal adhesions (green). Courtesy of Carmen Laethem, Aerie Pharma




These are examples of customer images that were submitted to our annual image competition. We are so confident about the quality of our images, we publish them every year in a competition to all of our users. The image quality speaks for itself. You need excellent image quality to fully exploit the power of an imaging assay – all these images taken with the IN Cell Analyzer 1000. Compatible with a broad range of objectives and plate/slide formats. An illustration of the image quality that can be achieved as well as the ability to overlay and fuse composite images. Note to ability to combine fluorescence and transmitted light images – 96 well UV/Cy5/DIC

Данные

пользователей

на IN Cell Analyzer

Rat cardiac H9C2 cells stained for DNA (blue), actin

(red) and tubulin

(green). IN Cell 1000.

Courtesy of Connla

Edwards, Trinity College Dublin

Primary pig trabecular

meshwork cells stained for actin

(green), DNA (blue) and paxillin

focal adhesion points (red). IN Cell 1000

Courtesy of Carmen Laethem, Aerie Pharma

Human neural stem cells stained for neuronal TUJ-1 (green), astrocyte

GFAP (red) and DNA (blue). IN Cell 1000.

Courtesy of Kymmy

Lorrain, BrainCells

Inc., USA




These are examples of customer images that were submitted to our annual image competition. We are so confident about the quality of our images, we publish them every year in a competition to all of our users. The image quality speaks for itself. You need excellent image quality to fully exploit the power of an imaging assay – all these images taken with the IN Cell Analyzer 1000. If you would like more information about any of the customer images in these slides, please let me know and I will obtain further details for you. Compatible with a broad range of objectives and plate/slide formats. An illustration of the image quality that can be achieved as well as the ability to overlay and fuse composite images. Note to ability to combine fluorescence and transmitted light images – 96 well UV/Cy5/DIC

+ Этопозид

До

воздействия

Качество


–

это

основаФормирование

ядрышка Срезы

тканей(семенники

мыши)Структура

цитоскелета(Актин, тубулин)

GFP репортеры(AKT в

PM ruffles)

Активация

рецептора(CypHer

5E)Рост

нейритов




It is essential to have high quality images generated quickly. This slide shows example images across a diverse range of applications.

Спасибо за внимание!


лебедева

Documents

d deconvolved2

48e100 d

dna blue

actin green

brain cells

actin red

dna microarrayb

tubulin green